Probiotic and prebiotic compositions, and methods of use thereof for treatment and prevention of graft versus host disease

ABSTRACT

Probiotic compositions containing non-pathogenic microbial entities, e.g., bacterial or fungal entities, are described herein. The probiotic compositions may optionally contain or be used in conjunction with one or more prebiotics. Uses of the probiotic compositions to treat or prevent transplant disorders, e.g., graft-versus-host disease (GVHD), in a subject are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/952,892, filed Nov. 25, 2015, which, in turn, claims priority to U.S.Provisional Patent Application No. 62/084,536, filed Nov. 25, 2014; U.S.Provisional Patent Application No. 62/084,537, filed Nov. 25, 2014; U.S.Provisional Patent Application No. 62/084,540, filed Nov. 25, 2014; U.S.Provisional Patent Application No. 62/117,632, filed Feb. 18, 2015; U.S.Provisional Patent Application No. 62/117,637, filed Feb. 18, 2015; U.S.Provisional Patent Application No. 62/117,639, filed Feb. 18, 2015; U.S.Provisional Patent Application No. 62/162,562, filed May 15, 2015; andU.S. Provisional Patent Application No. 62/257,714, filed Nov. 19, 2015.The entire contents of each of the foregoing applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 24, 2017, isnamed 126383_01905_SL.txt and is 3.95 megabytes in size.

BACKGROUND

Graft versus host disease (GVHD) is a common and devastatingcomplication following a hematopoietic or tissue transplant and occursin approximately 50% of transplant recipients. Acute GVHD is a majorsource of morbidity and mortality following allogeneic hematopoieticcell transplantation. Approximately 25,000 allogeneic hematopoietic celltransplants (e.g., bone marrow, peripheral blood stem cell [PBSC], orcord blood transplants) are performed annually worldwide. Over time, thenumber of transplants from unrelated donors, and in the number ofallogeneic transplants for AML, ALL, MDS, and lymphomas, continues torise. There is also an increase in the number of allogeneictransplantants for non-malignant diseases, and an increase in the numberof transplant patients over 50 years of age. The global incidence ofacute GVHD ranges from 26%-34% in recipients of fully matched, siblingdonor grafts to 42%-52% in recipients of matched, unrelated donorgrafts. Evidence from the US suggests that incidence ranges from 30% inrecipients of fully histocompatible transplants to 60%-70% in recipientsof mismatched hematopoietic cells or hematopoietic cells from anunrelated donor. There is no FDA approved treatment for either acute orchronic GVHD. Treatment strategies for acute GVHD aim to reduce theimmune reaction of the donor T cells against host tissues and thereforeincludes immunosuppressive treatment like cyclosporine, high dosesteroids, and methotrexate. The standard therapy for de novo acute GVHDis high dose methylprednisolone, with expected response rates of18%-50%. For patients who develop steroid-refractory acute GVHD, thereis no standard of care therapy, and expected survival is less than 30%.Therefore, novel therapies are urgently needed for the treatment andprevention of GVHD.

SUMMARY OF THE INVENTION

Disclosed herein are therapeutic compositions containing probiotic,non-pathogenic bacterial populations and networks thereof, for theprevention, control, and treatment of transplant disorders andconditions, in particular diseases associated with graft versus hostdisease (GVHD). In some embodiments, the therapeutic compositionscontain prebiotics, e.g., carbohydrates, in conjunction with microbialpopulations and/or networks thereof. These compositions are advantageousin being suitable for safe administration to humans and other mammaliansubjects and are efficacious in numerous dysbiotic diseases, disordersand conditions and in general nutritional health.

In one aspect, the instant invention provides a method of increasing theduration of survival of a subject receiving a transplant, e.g., a bonemarrow transplant, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population, such that theduration of survival of the subject is increased.

In one embodiment of the foregoing aspect, the bacterial population is ahuman-derived bacterial population.

In one embodiment of the foregoing aspect, administration of theprobiotic composition reduces the likelihood that the subject willdevelop sepsis following the bone marrow transplant. In one embodimentof the foregoing aspect, administration of the probiotic compositionreduces the likelihood that the subject will develop graft versus hostdisease (GVHD) following the bone marrow transplant.

In one embodiment of the foregoing aspect, the probiotic composition isadministered to the subject prior to receiving the bone marrowtransplant. In one embodiment of the foregoing aspect, the probioticcomposition is administered to the subject after receiving the bonemarrow transplant. In one embodiment of the foregoing aspect, theprobiotic composition is administered to the subject concurrently withthe bone marrow transplant.

In one embodiment of the foregoing aspect, the probiotic compositionreduces intestinal permeability in the subject.

In one embodiment of the foregoing aspect, the probiotic compositioncomprises a bacterial population that produces short chain fatty acids.In one embodiment of the foregoing aspect, the bacterial populationproduces a short chain fatty acid selected from the group consisting ofbutyrate, acetate, propionate, valerate, and combinations thereof.

In one embodiment of the foregoing aspect, the probiotic compositionreduces inflammation in the gastrointestinal tract of the subject. Inone embodiment of the foregoing aspect, the probiotic compositioncomprises an anti-inflammatory bacterial population. In one embodimentof the foregoing aspect, the anti-inflammatory bacterial populationdecreases secretion of pro-inflammatory cytokines and/or increasessecretion of anti-inflammatory cytokines by human peripheral bloodmononuclear cells (PBMCs). In one embodiment of the foregoing aspect,the anti-inflammatory bacterial population decreases secretion of apro-inflammatory cytokine selected from the group consisting of IFNγ,IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinationsthereof. In one embodiment of the foregoing aspect, theanti-inflammatory bacterial population increases secretion of ananti-inflammatory cytokine selected from the group consisting of IL-10,IL-13, IL-4, IL-5, TGFβ and combinations thereof. In one embodiment ofthe foregoing aspect, the anti-inflammatory bacterial populationproduces short chain fatty acids.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation comprises one or more bacterial species of the orderClostridiales. In one embodiment of the foregoing aspect, the bacterialspecies is from the genus Blautia, Clostridium, or Ruminococcus. In oneembodiment of the foregoing aspect, the bacterial population comprises asingle bacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises two or morebacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises a single bacterialspecies set forth in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E,or Table 1F. In one embodiment, the bacterial population comprises asingle bacterial species set forth in Table 5. In one embodiment of theforegoing aspect, the bacterial population comprises two or morebacterial species set forth in Table 1A, Table 1B, Table 1C, Table 1D,Table 1E, or Table 1F. In another embodiment, the bacterial populationcomprises two or more bacterial species set forth in Table 5.

In one embodiment of the foregoing aspect, the subject has a disorderselected from the group consisting of a hematopoietic neoplasticdisorder, leukemia, lymphoma, and multiple myeloma. In one embodiment ofthe foregoing aspect, the probiotic composition does not significantlyreduce or eliminate the graft versus tumor (GVT) effect of the bonemarrow transplant. In one embodiment of the foregoing aspect, thesubject has an autoimmune disorder. In one embodiment of the foregoingaspect, the autoimmune disorder is selected from the group consisting oflupus, multiple sclerosis, systemic sclerosis, Crohn's disease, type Idiabetes, and juvenile idiopathic arthritis. In one embodiment of theforegoing aspect, the subject has sickle cell disease or sickle cellanemia.

In embodiments of the foregoing aspects, the methods further compriseadministering a prebiotic to the subject. In one embodiment of theforegoing aspect, the prebiotic comprises a monomer or polymer selectedfrom the group consisting of arabinoxylan, xylose, soluble fiberdextran, soluble corn fiber, polydextrose, lactose,N-acetyl-lactosamine, glucose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises a monomer orpolymer selected from the group consisting of galactose, fructose,rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′sialylactose,6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, andcombinations thereof. In one embodiment of the foregoing aspect, theprebiotic comprises a monosaccharide selected from the group consistingof arabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, and combinations thereof. In one embodimentof the foregoing aspect, the prebiotic comprises a disaccharide selectedfrom the group consisting of xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises apolysaccharide, wherein the polysaccharide is xylooligosaccharide. Inone embodiment of the foregoing aspect, the prebiotic comprises a sugarselected from the group consisting of arabinose, fructose, fucose,lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose,xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose,xylooligosaccharide, and combinations thereof.

In another aspect, the instant invention provides a method of increasingthe duration of survival of a subject receiving a bone marrowtransplant, comprising administering to the subject a probioticcomposition comprising an isolated population of anti-inflammatorybacteria capable of decreasing secretion of pro-inflammatory cytokinesand/or increasing secretion of anti-inflammatory cytokines by humanperipheral blood mononuclear cells (PBMCs), and a pharmaceuticallyacceptable excipient, in an amount effective to reduce inflammation inthe gastrointestinal tract of the subject, such that the duration ofsurvival of the subject is increased.

In one embodiment of the foregoing aspect, the anti-inflammatorybacteria decrease secretion of pro-inflammatory cytokines and/orincrease secretion of anti-inflammatory cytokines by human peripheralblood mononuclear cells (PBMCs) in vitro.

In one embodiment of the foregoing aspect, the bacterial populationcomprises a single bacterial species set forth in Table 1. In oneembodiment of the foregoing aspect, the bacterial population comprisestwo or more bacterial species set forth in Table 1. In one embodiment ofthe foregoing aspect, the bacterial population comprises a singlebacterial species set forth in Table 1A, Table 1B, Table 1C, Table 1D,Table 1E, or Table 1F. In one embodiment of the foregoing aspect, thebacterial population comprises two or more bacterial species set forthin Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F.

In another aspect, the instant invention provides a method of increasingthe duration of survival of a subject receiving a bone marrowtransplant, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population, wherein theprobiotic composition reduces intestinal permeability in the subject;and administering to the subject a prebiotic that enhances the activityof the bacterial population, such that the duration of survival of thesubject is increased.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation produces short chain fatty acids. In one embodiment of theforegoing aspect, the bacterial population produces a short chain fattyacid selected from the group consisting of butyrate, acetate,propionate, valerate, and combinations thereof.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation comprises one or more bacterial species of the orderClostridiales. In one embodiment of the foregoing aspect, the bacterialspecies is from the genus Blautia, Clostridium, or Ruminococcus. In oneembodiment of the foregoing aspect, the bacterial population comprises asingle bacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises two or morebacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises a single bacterialspecies set forth in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E,or Table 1F. In one embodiment of the foregoing aspect, the bacterialpopulation comprises two or more bacterial species set forth in Table1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F.

In one embodiment of the foregoing aspect, the subject has a disorderselected from the group consisting of a hematopoietic neoplasticdisorder, leukemia, lymphoma, and multiple myeloma. In one embodiment ofthe foregoing aspect, the probiotic composition does not significantlyreduce or eliminate the graft versus tumor (GVT) effect of the bonemarrow transplant.

In one embodiment of the foregoing aspect, the subject has an autoimmunedisorder. In one embodiment of the foregoing aspect, the autoimmunedisorder is selected from the group consisting of lupus, multiplesclerosis, systemic sclerosis, Crohn's disease, type I diabetes, andjuvenile idiopathic arthritis.

In one embodiment of the foregoing aspect, the subject has sickle celldisease. In one embodiment of the foregoing aspect, the subject hassickle cell anemia.

In one embodiment of the foregoing aspect, the prebiotic comprises amonomer or polymer selected from the group consisting of arabinoxylan,xylose, soluble fiber dextran, soluble corn fiber, polydextrose,lactose, N-acetyl-lactosamine, glucose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises a monomer orpolymer selected from the group consisting of galactose, fructose,rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′sialylactose,6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, andcombinations thereof. In one embodiment of the foregoing aspect, theprebiotic comprises a monosaccharide selected from the group consistingof arabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, and combinations thereof. In one embodimentof the foregoing aspect, the prebiotic comprises a disaccharide selectedfrom the group consisting of xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises apolysaccharide, wherein the polysaccharide is xylooligosaccharide. Inone embodiment of the foregoing aspect, the prebiotic comprises a sugarselected from the group consisting of arabinose, fructose, fucose,lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose,xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose,xylooligosaccharide, and combinations thereof.

In another aspect, the instant invention provides a method of preventingor treating graft versus host disease (GVHD) in a subject receiving atransplant, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population, such that GVHDis prevented or treated.

In one embodiment of the foregoing aspect, the bacterial population is ahuman-derived bacterial population.

In one embodiment of the foregoing aspect, the subject is receiving ahematopoietic stem cell transplant. In one embodiment of the foregoingaspect, the subject is receiving a bone marrow transplant. In oneembodiment of the foregoing aspect, the subject is receiving a solidorgan transplant. In one embodiment of the foregoing aspect, the solidorgan transplant is selected from the group consisting of a kidneytransplant, a heart transplant, a lung transplant, a skin transplant, aliver transplant, a pancreas transplant, an intestinal transplant, anendocrine gland transplant, a bladder transplant, and a skeletal muscletransplant.

In one embodiment of the foregoing aspect, the subject has a disorderselected from the group consisting of a hematopoietic neoplasticdisorder, leukemia, lymphoma, and multiple myeloma. In one embodiment ofthe foregoing aspect, the probiotic composition does not significantlyreduce or eliminate the graft versus tumor (GVT) effect of the bonemarrow transplant.

In one embodiment of the foregoing aspect, the subject has an autoimmunedisorder. In one embodiment of the foregoing aspect, the autoimmunedisorder is selected from the group consisting of lupus, multiplesclerosis, systemic sclerosis, Crohn's disease, type I diabetes, andjuvenile idiopathic arthritis.

In one embodiment of the foregoing aspect, the subject has sickle celldisease or sickle cell anemia.

In one embodiment of the foregoing aspect, the probiotic compositionreduces intestinal permeability in the subject. In one embodiment of theforegoing aspect, the probiotic composition comprises a bacterialpopulation that produces short chain fatty acids. In one embodiment ofthe foregoing aspect, the bacterial population produces a short chainfatty acid selected from the group consisting of butyrate, acetate,propionate, valerate, and combinations thereof.

In one embodiment of the foregoing aspect, the probiotic compositionreduces inflammation in the gastrointestinal tract of the subject. Inone embodiment of the foregoing aspect, the probiotic compositioncomprises an anti-inflammatory bacterial population. In one embodimentof the foregoing aspect, the anti-inflammatory bacterial populationdecreases secretion of pro-inflammatory cytokines and/or increasessecretion of anti-inflammatory cytokines by human peripheral bloodmononuclear cells (PBMCs). In one embodiment of the foregoing aspect,the anti-inflammatory bacterial population decreases secretion of apro-inflammatory cytokine selected from the group consisting of IFNγ,IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinationsthereof. In one embodiment of the foregoing aspect, theanti-inflammatory bacterial population increases secretion of ananti-inflammatory cytokine selected from the group consisting of IL-10,IL-13, IL-4, IL-5, and combinations thereof. In one embodiment of theforegoing aspect, the anti-inflammatory bacterial population producesshort chain fatty acids.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation comprises one or more bacterial species of the orderClostridiales. In one embodiment of the foregoing aspect, the bacterialspecies is from the genus Blautia, Clostridium, or Ruminococcus. In oneembodiment of the foregoing aspect, the bacterial population comprises asingle bacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises two or morebacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises a single bacterialspecies set forth in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E,or Table 1F. In one embodiment of the foregoing aspect, the bacterialpopulation comprises two or more bacterial species set forth in Table1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F.

In embodiments of the foregoing aspects, the methods further compriseadministering a prebiotic to the subject. In one embodiment of theforegoing aspect, the prebiotic comprises a monomer or polymer selectedfrom the group consisting of arabinoxylan, xylose, soluble fiberdextran, soluble corn fiber, polydextrose, lactose,N-acetyl-lactosamine, glucose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises a monomer orpolymer selected from the group consisting of galactose, fructose,rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′sialylactose,6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, andcombinations thereof. In one embodiment of the foregoing aspect, theprebiotic comprises a monosaccharide selected from the group consistingof arabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, and combinations thereof. In one embodimentof the foregoing aspect, the prebiotic comprises a disaccharide selectedfrom the group consisting of xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises apolysaccharide, wherein the polysaccharide is xylooligosaccharide. Inone embodiment of the foregoing aspect, the prebiotic comprises a sugarselected from the group consisting of arabinose, fructose, fucose,lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose,xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose,xylooligosaccharide, and combinations thereof.

In another aspect, the instant invention provides a method of reducinginflammation in the gastrointestinal tract of a subject receiving atransplant, comprising administering to the subject a probioticcomposition comprising an isolated, anti-inflammatory bacterialpopulation and a pharmaceutically acceptable excipient, such thatinflammation in the gastrointestinal tract of the subject receiving thetransplant is reduced.

In one embodiment of the foregoing aspect, the anti-inflammatorybacterial population decreases secretion of pro-inflammatory cytokinesand/or increases secretion of anti-inflammatory cytokines by humanperipheral blood mononuclear cells (PBMCs). In one embodiment of theforegoing aspect, the anti-inflammatory bacterial population decreasessecretion of a pro-inflammatory cytokine selected from the groupconsisting of IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β,TNFα, and combinations thereof. In one embodiment of the foregoingaspect, the anti-inflammatory bacterial population increases secretionof an anti-inflammatory cytokine selected from the group consisting ofIL-10, IL-13, IL-4, IL-5, TGFβ, and combinations thereof. In oneembodiment of the foregoing aspect, the anti-inflammatory bacterialpopulation produces short chain fatty acids.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation comprises one or more bacterial species of the orderClostridiales. In one embodiment of the foregoing aspect, the bacterialspecies is from the genus Blautia, Clostridium, or Ruminococcus. In oneembodiment of the foregoing aspect, the bacterial population comprises asingle bacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises two or morebacterial species set forth in Table 1. In one embodiment of theforegoing aspect, the bacterial population comprises a single bacterialspecies set forth in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E,or Table 1F. In one embodiment of the foregoing aspect, the bacterialpopulation comprises two or more bacterial species set forth in Table1A, Table 1B, Table 1C, Table 1D, Table 1E, or Table 1F.

In embodiments of the foregoing aspects, the methods further comprisingadministering a prebiotic to the subject. In one embodiment of theforegoing aspect, the prebiotic comprises a monomer or polymer selectedfrom the group consisting of arabinoxylan, xylose, soluble fiberdextran, soluble corn fiber, polydextrose, lactose,N-acetyl-lactosamine, glucose, and combinations thereof. In oneembodiment of the foregoing aspect, wherein the prebiotic comprises amonomer or polymer selected from the group consisting of galactose,fructose, rhamnose, mannose, uronic acids, 3′-fucosyllactose,3′sialylactose, 6′-sialyllactose, lacto-N-neotetraose,2′-2′-fucosyllactose, and combinations thereof. In one embodiment of theforegoing aspect, the prebiotic comprises a monosaccharide selected fromthe group consisting of arabinose, fructose, fucose, lactose, galactose,glucose, mannose, D-xylose, xylitol, ribose, and combinations thereof.In one embodiment of the foregoing aspect, the prebiotic comprises adisaccharide selected from the group consisting of xylobiose, sucrose,maltose, lactose, lactulose, trehalose, cellobiose, and combinationsthereof. In one embodiment of the foregoing aspect, the prebioticcomprises a polysaccharide, wherein the polysaccharide isxylooligosaccharide. In one embodiment of the foregoing aspect, theprebiotic comprises a sugar selected from the group consisting ofarabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, xylooligosaccharide, and combinationsthereof.

In another aspect, the instant invention provides a method of reducingintestinal permeability in a subject receiving a transplant, comprisingadministering to the subject a probiotic composition comprising anisolated bacterial population and a pharmaceutically acceptableexcipient, such that the intestinal permeability of the subject of thesubject receiving the transplant is reduced.

In one embodiment of the foregoing aspect, the isolated bacterialpopulation produces short chain fatty acids. In one embodiment of theforegoing aspect, the bacterial population produces a short chain fattyacid selected from the group consisting of butyrate, acetate,propionate, valerate, and combinations thereof. In one embodiment of theforegoing aspect, the bacterial population produces butyrate.

In one embodiment of the foregoing aspect, the method further comprisesadministering a prebiotic to the subject. In one embodiment of theforegoing aspect, the prebiotic comprises a monomer or polymer selectedfrom the group consisting of arabinoxylan, xylose, soluble fiberdextran, soluble corn fiber, polydextrose, lactose,N-acetyl-lactosamine, glucose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises a monomer orpolymer selected from the group consisting of galactose, fructose,rhamnose, mannose, uronic acids, 3′-fucosyllactose, 3′sialylactose,6′-sialyllactose, lacto-N-neotetraose, 2′-2′-fucosyllactose, andcombinations thereof. In one embodiment of the foregoing aspect, theprebiotic comprises a monosaccharide selected from the group consistingof arabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, and combinations thereof. In one embodimentof the foregoing aspect, the prebiotic comprises a disaccharide selectedfrom the group consisting of xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, and combinations thereof. In oneembodiment of the foregoing aspect, the prebiotic comprises apolysaccharide, wherein the polysaccharide is xylooligosaccharide. Inone embodiment of the foregoing aspect, the prebiotic comprises a sugarselected from the group consisting of arabinose, fructose, fucose,lactose, galactose, glucose, mannose, D-xylose, xylitol, ribose,xylobiose, sucrose, maltose, lactose, lactulose, trehalose, cellobiose,xylooligosaccharide, and combinations thereof.

In one aspect, the instant invention provides a method of increasing theduration of survival of a subject receiving a bone marrow transplant,comprising administering to the subject a probiotic compositioncomprising an isolated, human-derived bacterial population, such thatthe duration of survival of the subject is increased.

In one embodiment of the foregoing aspect, administration of theprobiotic composition reduces the likelihood that the subject willdevelop sepsis following the bone marrow transplant. In one embodimentof the foregoing aspect, administration of the probiotic compositionreduces the likelihood that the subject will develop graft versus hostdisease (GVHD) following the bone marrow transplant.

In another aspect, the instant invention provides a method of increasingthe duration of survival of a subject receiving a bone marrowtransplant, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population and apharmaceutically acceptable excipient, wherein the probiotic compositionreduces intestinal permeability in the subject; and administering to thesubject a prebiotic that enhances the activity of the bacterialpopulation, such that the duration of survival of the subject isincreased.

In another aspect, the instant invention provides a method of preventinggraft versus host disease (GVHD) in a subject receiving a transplant,comprising administering to the subject a probiotic compositioncomprising an isolated, human-derived bacterial population, such thatGVHD is prevented.

In some embodiments of the foregoing aspects, the subject is receiving ahematopoietic stem cell transplant. In some embodiments of the foregoingaspects, the subject is receiving a bone marrow transplant. In someembodiments of the foregoing aspects, the subject is receiving a solidorgan transplant. In some embodiments of the foregoing aspects, thesolid organ transplant is selected from the group consisting of a kidneytransplant, a heart transplant, a lung transplant, a skin transplant, aliver transplant, a pancreas transplant, an intestinal transplant, anendocrine gland transplant, a bladder transplant, and a skeletal muscletransplant.

In another aspect, the instant invention provides a method of reducinginflammation in the gastrointestinal tract of a subject receiving atransplant, comprising administering to the subject a probioticcomposition comprising an isolated, anti-inflammatory bacterialpopulation, such that inflammation in the gastrointestinal tract of thesubject receiving the transplant is reduced.

In another aspect, the instant invention provides a method of reducingintestinal permeability in a subject receiving a transplant, comprisingadministering to the subject a probiotic composition comprising anisolated bacterial population, such that the intestinal permeability ofthe subject receiving the transplant is reduced.

In another aspect, the instant invention provides a pharmaceuticalcomposition comprising an isolated anti-inflammatory bacterialpopulation capable of decreasing secretion of a pro-inflammatorycytokine and/or increasing secretion of an anti-inflammatory cytokine byhuman peripheral blood mononuclear cells (PBMCs), and a pharmaceuticallyacceptable excipient.

In one embodiment of the foregoing aspect, the composition furthercomprising a prebiotic.

In some embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Acidaminococcus intestine. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Acinetobacter baumannii. In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Acinetobacter lwoffii. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Akkermansia muciniphila. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Alistipes putredinis. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofAlistipes shahii. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Anaerostipeshadrus. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Anaerotruncuscolihominis. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Bacteroides caccae. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Bacteroides cellulosilyticus. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Bacteroides dorei. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bacteroides eggerthii. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Bacteroides finegoldii. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Bacteroides fragilis. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofBacteroides massiliensis. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population of Bacteroidesovatus. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Bacteroides salanitronis.In some embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Bacteroides salyersiae. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bacteroides sp. 1_1_6. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Bacteroides sp. 3_1_23. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Bacteroides sp. D20. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofBacteroides thetaiotaomicron. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofBacteroides uniformis. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Bacteroidesvulgatus. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Bifidobacteriumadolescentis. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Bifidobacteriumbifidum. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Bifidobacterium breve. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Bifidobacterium faecale. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bifidobacterium kashiwanohense. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bifidobacterium longum subsp. longum. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bifidobacterium pseudocatenulatum. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Bifidobacterium stercoris. In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Blautia (Ruminococcus) coccoides. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Blautia faecis. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofBlautia glucerasea. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Blautia(Ruminococcus) hansenii. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population of Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus). In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Blautia (Ruminococcus) luti. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Blautia (Ruminococcus) obeum. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Blautia producta (Ruminococcus productus). In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Blautia (Ruminococcus) schinkii. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Blautia stercoris. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Blautia unculturedbacterium clone SJTU_B_14_30 (GenBank: EF402926.1). In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Blautia wexlerae. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Candidatus Arthromitus sp. SFB-mouse-Yit. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Catenibacterium mitsuokai. In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Clostridiaceae bacterium (Dielma fastidiosa) JC13. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Clostridiales bacterium 1_7_47FAA. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Clostridium asparagiforme. In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Clostridium bolteae. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofClostridium clostridioforme. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofClostridium glycyrrhizinilyticum. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofClostridium (Hungatella) hathewayi. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofClostridium histolyticum. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population of Clostridiumindolis. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Clostridium leptum. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Clostridium (Tyzzerella) nexile. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Clostridium perfringens. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Clostridium (Erysipelatoclostridium) ramosum.In some embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Clostridium scindens. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Clostridium sp. 14774. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Clostridium sp. 7_3_54FAA. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Clostridium sp. HGF2. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofClostridium symbiosum. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Collinsellaaerofaciens. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Collinsella intestinalis.In some embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Coprobacillus sp. D7. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Coprococcus catus. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Coprococcus comes. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofDorea formicigenerans. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Dorealongicatena. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Enterococcus faecalis. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Enterococcus faecium. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Erysipelotrichaceae bacterium 3_1_53. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Escherichia coli. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Escherichia coli S88. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofEubacterium eligens. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Eubacteriumfissicatena. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Eubacterium ramulus. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Eubacterium rectale. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Faecalibacterium prausnitzii. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Flavonifractor plautii. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Fusobacterium mortiferum. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Fusobacterium nucleatum. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Holdemania filiformis. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Hydrogenoanaerobacterium saccharovorans. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Klebsiella oxytoca. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Lachnospiraceae bacterium 3_1_57FAA_CT1. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Lachnospiraceae bacterium 7_1_58FAA. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Lachnospiraceae bacterium 5_1_57FAA. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Lactobacillus casei. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Lactobacillus rhamnosus. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Lactobacillus ruminis. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Lactococcus casei. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofOdoribacter splanchnicus. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population ofOscillibacter valericigenes. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofParabacteroides gordonii. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population ofParabacteroides johnsonii. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population ofParabacteroides merdae. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population of Pediococcusacidilactici. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population ofPeptostreptococcus asaccharolyticus. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Propionibacterium granulosum. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Roseburia intestinalis. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Roseburia inulinivorans. In some embodiments of theforegoing aspects, the bacterial population comprises an isolatedpopulation of Ruminococcus faecis. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofRuminococcus gnavus. In some embodiments of the foregoing aspects, thebacterial population comprises an isolated population of Ruminococcussp. ID8. In some embodiments of the foregoing aspects, the bacterialpopulation comprises an isolated population of Ruminococcus torques. Insome embodiments of the foregoing aspects, the bacterial populationcomprises an isolated population of Slackia piriformis. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Staphylococcus epidermidis. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Staphylococcus saprophyticus. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Streptococcus cristatus. In some embodimentsof the foregoing aspects, the bacterial population comprises an isolatedpopulation of Streptococcus dysgalactiae subsp. equisimilis. In someembodiments of the foregoing aspects, the bacterial population comprisesan isolated population of Streptococcus infantis. In some embodiments ofthe foregoing aspects, the bacterial population comprises an isolatedpopulation of Streptococcus oralis. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofStreptococcus sanguinis. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population ofStreptococcus viridans. In some embodiments of the foregoing aspects,the bacterial population comprises an isolated population ofStreptococcus thermophiles. In some embodiments of the foregoingaspects, the bacterial population comprises an isolated population ofVeillonella dispar.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides a list of Operational Taxonomic Units (OTU) withtaxonomic assignments made to Genus, Species, and Phylogenetic Clade.Clade membership of bacterial OTUs is based on 16S sequence data. Cladesare defined based on the topology of a phylogenetic tree that isconstructed from full-length 16S sequences using maximum likelihoodmethods familiar to individuals with ordinary skill in the art ofphylogenetics. Clades are constructed to ensure that all OTUs in a givenclade are: (i) within a specified number of bootstrap supported nodesfrom one another, and (ii) within 5% genetic similarity. OTUs that arewithin the same clade can be distinguished as genetically andphylogenetically distinct from OTUs in a different clade based on 16S-V4sequence data, while OTUs falling within the same clade are closelyrelated. OTUs falling within the same clade are evolutionarily closelyrelated and may or may not be distinguishable from one another using16S-V4 sequence data. Members of the same clade, due to theirevolutionary relatedness, play similar functional roles in a microbialecology such as that found in the human gut. Compositions substitutingone species with another from the same clade are likely to haveconserved ecological function and therefore are useful in the presentinvention. All OTUs are denoted as to their putative capacity to formspores and whether they are a Pathogen or Pathobiont (see Definitionsfor description of “Pathobiont”). NIAID Priority Pathogens are denotedas ‘Category-A’, ‘Category-B’, or ‘Category-C’, and OpportunisticPathogens are denoted as ‘OP’. OTUs that are not pathogenic or for whichtheir ability to exist as a pathogen is unknown are denoted as ‘N’. The‘SEQ ID Number’ denotes the identifier of the OTU in the SequenceListing File and ‘Public DB Accession’ denotes the identifier of the OTUin a public sequence repository. See, e.g., WO2014/121304.

Table 1A provides a list of exemplary bacteria useful in the presentinvention.

Table 1B provides a list of exemplary bacteria useful in the presentinvention.

Table 1C provides a list of exemplary bacteria useful in the presentinvention.

Table 1D provides a list of exemplary bacteria useful in the presentinvention.

Table 1E provides a list of exemplary bacteria useful in the presentinvention. These bacteria are preferably down-modulated in a subject.

Table 1F provides a list of exemplary bacteria that may be used in theinvention. These bacteria are preferably up-modulated in a subject.

Table 2A lists species identified as “germinable” and “sporulatable” bycolony picking approach.

Table 2B lists species identified as “germinable” using 16S colonypicking approach.

Table 2C lists species identified as “sporulatable” using 16s-V4 NGSapproach. See, e.g., WO2014/121304.

Table 3 provides criteria for stages of acute GVHD.

Table 4 provides representative examples of microbial enzymes that allowutilization of prebiotics.

Table 5 provides a list of species enriched in alive GVHD patients.

Table 6 lists anaerobic bacterial species tested for carbon sourceusage.

Table 7 provides exemplary prebiotics/carbon sources for use in thecompositions and methods of the invention.

Table 8 provides bacterial species detected at low frequency in vaginalsamples from vancomycin-treated mice (day 6) that were not present inuntreated mice (day 0).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting serum endotoxin levels (EU/ml) over timefollowing treatment with xylose. Treatment of mice with xylose alonereduces basal levels of serum endotoxin (day 14 vs day 0). Antibiotictreatment (Ciprofloxacin (cipro) or enrofloxacin (enro)) leads to anincrease in serum endotoxin levels (measured 2 days after a 5 daycourse, at day 0) with a return to baseline by day 14. Xylosecounteracts the endotoxin increase caused by cipro but not enroantibiotic treatment.

FIG. 2a -FIG. 2o is a panel of graphs showing the time course of Th1related cytokines that were released by human peripheral mononuclearcells (PBMCs) incubated with Ruminococcus gnavus (Epv 1), Eubacteriumrectale (Epv 2), Blautia luti (Epv 3), Blautia wexlerae (Epv 5) andEnterococcus faecalis (Epv 8), or combinations of each bacterium with E.faecalis. Amounts of interferon gamma (IFN-γ), IL-12p70, IL-6, IL-2 andTNFα that were released in culture supernatants by PBMCs were measuredafter 24, 48 and 72 hours. a) IFN-γ concentration (pg/ml) after 24hours. b) IFN-γ concentration (pg/ml) after 48 hours. c) IFN-γconcentration (pg/ml) after 72 hours. d) IL-12p70 concentration (pg/ml)after 24 hours. e) IL-12p70 concentration (pg/ml) after 48 hours. f)IL-12p70 concentration (pg/ml) after 72 hours. g) IL-6 concentration(pg/ml) after 24 hours. h) IL-6 concentration (pg/ml) after 48 hours. i)IL-6 concentration (pg/ml) after 72 hours. j) IL-2 concentration (pg/ml)after 24 hours. k) IL-2 concentration (pg/ml) after 48 hours. 1) IL-2concentration (pg/ml) after 72 hours. m) TNFα concentration (pg/ml)after 24 hours. n) TNFα concentration (pg/ml) after 48 hours. o) TNFαconcentration (pg/ml) after 72 hours.

FIG. 3a -FIG. 3i is a panel of graphs showing the time course of Th2related cytokines that were released by human PBMCs incubated with R.gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5)and E. faecalis (Epv 8), or combinations of each bacterium with E.faecalis. Amounts of IL-13, IL-4 and IL-5 that were released in culturesupernatants by PBMCs were measured after 24, 48 and 72 hours. a) IL-13concentration (pg/ml) after 24 hours. b) IL-13 concentration (pg/ml)after 48 hours. c) IL-13 concentration (pg/ml) after 72 hours. d) IL-4concentration (pg/ml) after 24 hours. e) IL-4 concentration (pg/ml)after 48 hours. f) IL-4 concentration (pg/ml) after 72 hours. g) IL-5concentration (pg/ml) after 24 hours. h) IL-5 concentration (pg/ml)after 48 hours. i) IL-5 concentration (pg/ml) after 72 hours.

FIG. 4a -FIG. 4i is a panel of graphs showing the time course of Th9,Th17 and Treg cytokines that were released by human PBMCs incubated withR. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv5) and E. faecalis (Epv 8), or combinations of each bacterium with E.faecalis. Amounts of IL-9, IL-17 and IL-10 that were released in culturesupernatants by PBMCs were measured after 24, 48 and 72 hours. a) IL-9concentration (pg/ml) after 24 hours. b) IL-9 concentration (pg/ml)after 48 hours. c) IL-9 concentration (pg/ml) after 72 hours. d) IL-17concentration (pg/ml) after 24 hours. e) IL-17 concentration (pg/ml)after 48 hours. f) IL-17 concentration (pg/ml) after 72 hours. g) IL-10concentration (pg/ml) after 24 hours. h) IL-10 concentration (pg/ml)after 48 hours. i) IL-10 concentration (pg/ml) after 72 hours.

FIG. 5a -FIG. 5x is a panel of graphs showing the time course ofmonocyte, macrophage and neutrophil-derived inflammatory cytokines thatwere released by human PBMCs incubated with R. gnavus (Epv 1), E.rectale (Epv 2), B. luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis(Epv 8), or combinations of each bacterium with E. faecalis. Amounts ofmonocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein113 (MIP1β), macrophage inflammatory protein 1α (MIP1α), regulated onactivation, normal T expressed and secreted protein (RANTES),interleukin-1α (IL-1α), interleukin-1β (IL1β), interferon α2 (IFN-α2)and interleukin-8 (IL-8) that were released in culture supernatants byPBMCs were measured after 24, 48 and 72 hours. a) MCP-1 concentration(pg/ml) after 24 hours. b) MCP-1 concentration (pg/ml) after 48 hours.c) MCP-1 concentration (pg/ml) after 72 hours. d) MIP1β concentration(pg/ml) after 24 hours. e) MIP1β concentration (pg/ml) after 48 hours.f) MIP1β concentration (pg/ml) after 72 hours. g) MIP1α concentration(pg/ml) after 24 hours. h) MIP1α concentration (pg/ml) after 48 hours.i) MIP1α concentration (pg/ml) after 72 hours. j) RANTES concentration(pg/ml) after 24 hours. k) RANTES concentration (pg/ml) after 48hours. 1) RANTES concentration (pg/ml) after 72 hours. m) IL-1αconcentration (pg/ml) after 24 hours. n) IL-1α concentration (pg/ml)after 48 hours. o) IL-1α concentration (pg/ml) after 72 hours. p) IL1βconcentration (pg/ml) after 24 hours. q) IL1β concentration (pg/ml)after 48 hours. r) IL1β concentration (pg/ml) after 72 hours. s) IFN-α2concentration (pg/ml) after 24 hours. t) IFN-α2 concentration (pg/ml)after 48 hours. u) IFN-α2 concentration (pg/ml) after 72 hours. v) IL-8concentration (pg/ml) after 24 hours. w) IL-8 concentration (pg/ml)after 48 hours. x) IL-8 concentration (pg/ml) after 72 hours.

FIG. 6a -FIG. 6d is a panel of graphs showing the secreted levels ofcytokines IFNγ (Ifng), IL-12p70, IL-1α (IL-1a), IL-6, IL-8, MCP1, MIP1α(MIP1α), MIP1β (MIP1b), TNFα (TNFa), IL-10, IL-13, IL-9, IL-4, IL-5,IL-17α (IL-17A) and IL-2 produced by PBMCs in the presence of a) R.gnavus, b) B. wexlerae, c) E. rectale and d) B. luti, alone or incombination with E. faecalis (Epv 8), relative to levels secretedfollowing treatment with E. faecalis alone for 24 hours (E.faecalis=100%).

FIG. 7a -FIG. 7p is a panel of graphs that show the effect of R. gnavus(Epv1) on cytokine concentration (pg/ml) either alone or in combinationwith Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml).a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8,h) IL17A, i) IL-α, j) IL-9, k) IL-2, 1) IL-4, m) IL-5, n) MIP-1α, o)MIP-1β, p) TNF-α.

FIG. 8a -FIG. 8p is a panel of graphs that show the effect of E. rectale(Epv2) on cytokine concentration (pg/ml) either alone or in combinationwith Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml).a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8,h) IL17A, i) IL-α, j) IL-9, k) IL-2, 1) IL-4, m) IL-5, n) MIP-1α, o)MIP-1β, p) TNF-α.

FIG. 9a -FIG. 9p is a panel of graphs that show the effect of B. luti(Epv3) on cytokine concentration (pg/ml) either alone or in combinationwith Epv 8 (E. faecalis) on cytokine production by human PBMCs (pg/ml).a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f) MCP-1, g) IL-8,h) IL17α, i) IL-α, j) IL-9, k) IL-2, 1) IL-4, m) IL-5, n) MIP-1α, o)MIP-1β, p) TNF-α.

FIG. 10a -FIG. 10p is a panel of graphs that show the effect of B.wexlarae) on cytokine concentration (pg/ml) either alone or incombination with Epv 8 (E. faecalis) on cytokine production by humanPBMCs (pg/ml). a) IL-6, b) IFN-γ, c) IL-13, d) IL-10, e) IL-12p70, f)MCP-1, g) IL-8, h) IL17α, i) IL-α, j) IL-9, k) IL-2, 1) IL-4, m) IL-5,n) MIP-1α, o) MIP-1β, p) TNF-α.

FIG. 11a -FIG. 11d is a panel of graphs showing that (a-b) EPV3 iscapable of inducing a desirable anti-inflammatory cytokine profile fortreating or preventing GVHD and (c-d) EPV5 induces a suboptimal profilefor GVHD.

FIG. 12a -FIG. 12b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv6(Clostridium leptum).

FIG. 13a -FIG. 13b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv15 (Blautiafaecis).

FIG. 14a -FIG. 14b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv20(Blautia/Ruminococcus obeum ATCC 29174).

FIG. 15a -FIG. 15b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv21 (Blautiaproducta ATCC 27340).

FIG. 16a -FIG. 16b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv22 (Blautiacoccoides ATCC 29236).

FIG. 17a -FIG. 17b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv23 (Blautiahydrogenotrophica ATCC BAA-2371).

FIG. 18a -FIG. 18b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv24 (BlautiaHansenii ATCC27752).

FIG. 19a -FIG. 19b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv35(Eubacterium rectale).

FIG. 20a -FIG. 20b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv47(previously uncultured Blautia, similar to GQ898099_s S1-5).

FIG. 21a -FIG. 21b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv51(previously uncultured Blautia, similar to SJTU_C_14_16).

FIG. 22a -FIG. 22b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv52 (Blautiawexlerae (SJTU_B_09_77)).

FIG. 23a -FIG. 23b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv54 (Blautialuti ELU0087-T13-S—NI_000247).

FIG. 24a -FIG. 24b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv64 (Blautiawexlerae WAL 14507).

FIG. 25a -FIG. 25b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv78 (Blautiaobeum).

FIG. 26a -FIG. 26b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv102(Ruminococcus gnavus).

FIG. 27a -FIG. 27b depicts the production of (a) pro-inflammatory(IL-12p70, IFNγ, IP-10, IL-1RA) and (b) anti-inflammatory (IL-10, IL-4,IL-13) cytokines by human PBMCs following treatment with Epv114 (Blautialuti (BlnIX)).

FIG. 28a -FIG. 28d presents results from flow cytometry analysis of Tcell populations in human PBMCs incubated in the presence of variouscommensal bacteria, determined using flow cytometry. A) Proportion ofTreg cells (CD25⁺CD127^(lo)); B) Proportion of Th17 cells (CXCR3⁻CCR6⁺);C) Proportion of Th1 cells (CXCR3⁺CCR6⁻); D) Proportion of Th2 cells(CXCR3⁻CCR6⁻). Bacterial strains are as follows: Epv 1: R. gnavus; Epv3: B. luti; Epv 2: E. rectale; Epv 5: B. wexlerae; Epv. 8: E. faecalis;Epv 20: B. obeum; Epv 21: B. producta; Epv 24: B. hansenii. The resultsare shown as percent (%) of CD3ε⁺CD4⁺ cells.

FIG. 29a -FIG. 29u presents the preferred carbon sources utilized byvarious commensal bacteria. (a) R. gnavus; (b) E. rectale; (c) C.leptum; (d) B. luti; (e) B. wexlerae; (f) B. faecis; (g) B. obeum; (h)B. producta; (i) B. coccoides; (j) B. hydrogenotrophica; (k) B.hansenii; (1) B. luti Blnl X; (m) B. luti ELU; (n) R. gnavus; (o) B.faecis; (p) R. torques; (q) B. wexlerae WAL14507; (r) B. wexlerae SJTU,(s) SJTU1416; (t) GQ8980099; (u) E. rectale.

FIG. 30 graphically depicts levels of serum IFNγ before, during, andafter treatment with a prebiotic formulation containing xylose.

FIG. 31 is a graph that shows the change in Chao1 diversity (indicatorof community richness) over time in subjects administered xylose threetimes per day (TID) at 1, 2, 8, 12.5 or 15 grams.

FIG. 32 depicts the impact of oral vancomycin on the microbiome of thegut and the vagina, by principal component analysis (PCA).

DETAILED DESCRIPTION I. Overview

Disclosed herein are therapeutic compositions containing bacterialentities (e.g., non-pathogenic germination-competent bacterialentities), fungal entities, and/or prebiotics for the prevention,control, and treatment of immune and inflammatory diseases, disordersand conditions, and for general nutritional health. These compositionsare advantageous in being suitable for safe administration to humans andother mammalian subjects and are efficacious in treating or preventingnumerous immune and inflammatory diseases and gastrointestinal diseases,disorders and conditions associated with a dysbiosis.

While spore-based compositions are known, these are generally preparedaccording to various techniques such as lyophilization or spray-dryingof liquid bacterial cultures, resulting in poor efficacy, instability,substantial variability and lack of adequate safety and efficacy.

It has now been found that populations of bacterial entities can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and can be administered to mammalian subjects inaccordance with the methods described herein.

The microbes that inhabit the human gastrointestinal tract, skin, lungs,vagina, and other niches are starting to be understood and appreciatedfor their roles in human health and disease (e.g. see Human MicrobiomeProject Consortium 2012, Structure, function, and diversity of thehealthy human microbiome. Nature 486(7402):207-14). Aspects of theinvention are based, in part, on the realization that, althoughautoimmune and inflammatory diseases are often attributed to geneticmutations, these conditions are also influenced by microbes. It is alsoappreciated that, because microbes not only interact with the host butwith one another, the immunomodulatory behavior of microbes can dependon relationships between microbes. For example, a microbial network in agiven niche may comprise diverse microbes that all accomplish one ormore of the same functions, or may instead comprise diverse microbesthat all individually contribute to accomplish one or more functions. Inanother example, microbes in a given niche may compete with one anotherfor nutrients or space.

Microbes may influence the risk, progression, or treatment efficacy ofan autoimmune or inflammatory disease. In certain aspects, microbes playa role in the prevention of an autoimmune or inflammatory disease or inthe suppression of an innate or adaptive immune response. Conversely, incertain aspects, microbes may stimulate an inflammatory immune responseand thereby contribute to, increase the risk of, or worsen the symptomsof an autoimmune or inflammatory disease. In certain aspects, somemicrobes may be associated with lower disease severity or mortality.

Accordingly, disclosed herein are compositions and methods for theprevention and/or treatment of disorders associated with disruptions ofthe systemic microbiome, e.g., autoimmune and inflammatory diseases, inhuman subjects.

II. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, “a compound” includes mixtures ofcompounds.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 3 or more than 3 standard deviations,jper the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, or within5-fold, or within 2-fold, of a value.

As used herein, the term “purified bacterial preparation” refers to apreparation that includes “isolated” bacteria or bacteria that have beenseparated from at least one associated substance found in a sourcematerial or any material associated with the bacteria in any processused to produce the preparation.

A “bacterial entity” includes one or more bacteria. Generally, a firstbacterial entity is distinguishable from a second bacterial entity.

As used herein, the term “formation” refers to synthesis or production.

As used herein, the term “inducing” means increasing the amount oractivity of a given material as dictated by context.

As used herein, the term “depletion” refers to reduction in amount of.

As used herein, a “prebiotic” refers to an ingredient that allowsspecific changes, both in the composition and/or activity in thegastrointestinal microbiota that may (or may not) confer benefits uponthe host. In some embodiments, a prebiotic can be a comestible food orbeverage or ingredient thereof. In some embodiments, a prebiotic may bea selectively fermented ingredient. Prebiotics may include complexcarbohydrates, amino acids, peptides, minerals, or other essentialnutritional components for the survival of the bacterial composition.Prebiotics include, but are not limited to, amino acids, biotin,fructooligosaccharide, galactooligosaccharides, hemicelluloses (e.g.,arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin,lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums(e.g., guar gum, gum arabic and carregenaan), oligofructose,oligodextrose, tagatose, resistant maltodextrins (e.g., resistantstarch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan,citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers(e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber)and xylooligosaccharides.

As used herein, “predetermined ratios” refer to ratios determined orselected in advance.

As used herein, “germinable bacterial spores” are spores capable offorming vegetative cells in response to a particular cue (e.g., anenvironmental condition or a small molecule).

As used herein, “detectably present” refers to presence in an amountthat can be detected using assays provided herein or otherwise known inthe art that exist as of the filing date.

As used herein, “augmented” refers to an increase in amount and/orlocalization within to a point where it becomes detectably present.

As used herein, “fecal material” refers to a solid waste product ofdigested food and includes feces or bowel washes.

As used herein, the phrase “host cell response” refers to a responseproduced by a cell of a host organism.

As used herein, a “mammalian subject protein” refers to a proteinproduced by a mammalian subject and encoded by the mammalian subjectgenome. The term mammalian subject protein includes proteins that havebeen post-translationally processed and/or modified.

As used herein, the term “food-derived” refers to a protein orcarbohydrate found in a consumed food.

As used herein, the term “biological material” refers to a materialproduced by a biological organism.

As used herein, the term “detection moiety” refers to an assay componentthat functions to detect an analyte.

As used herein, the term “incomplete network” refers to a partialnetwork that lacks at least one of the entire set of components neededto carry out one or more network functions.

As used herein, the term “supplemental” refers to something that isadditional and non-identical.

As used herein, a composition is “substantially free” of microbes whenmicrobes are absent or undetectable as determined by the use of standardgenomic and microbiological techniques. A composition is “substantiallyfree” of a prebiotic or immunostimulatory carbohydrate whennon-microbial carbohydrates are absent or undetectable as determined bythe use of standard biochemical techniques, e.g., dye-based assays.

Microbial agents (individual or populations of microbes, microbialnetworks or parts of networks, or microbial metabolites) are consideredto be “exogenous” to a subject (e.g., a human or non-human animal), acell, tissue, organ or other environment of a human or non-human animal,if said subject, or said cell, tissue, organ or other environment of thesubject, does not contain detectable levels of the microbial agent.

A microbial agent or population thereof is “heterologous” or“heterologously contained” on or in a host environment when, e.g., themicrobial agent or population is administered or disposed on or in thehost or host environment in a number, concentration, form or othermodality that is not found in the host prior to administration of themicrobial agent or population, or when the microbial agent or populationcontains an activity or structural component different from a host thatdoes not naturally have the microbial agent within the targetenvironment to which the microbe is administered or thereafter disposed.

As used herein, the term “antioxidant” is understood to include any oneor more of various substances such as beta-carotene (a vitamin Aprecursor), vitamin C, vitamin E, and selenium) that inhibit oxidationor reactions promoted by Reactive Oxygen Species (“ROS”) and otherradical and non-radical species. Additionally, antioxidants aremolecules capable of slowing or preventing the oxidation of othermolecules. Non-limiting examples of antioxidants include astaxanthin,carotenoids, coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji(wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene,polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, orcombinations thereof.

“Backbone Network Ecology” or simply “Backbone Network” or “Backbone”are compositions of microbes that form a foundational composition thatcan be built upon or subtracted from to optimize a Network Ecology orFunctional Network Ecology to have specific biological characteristicsor to comprise desired functional properties, respectively. Microbiometherapeutics can be comprised of these “Backbone Networks Ecologies” intheir entirety, or the “Backbone Networks” can be modified by theaddition or subtraction of “R-Groups” to give the network ecologydesired characteristics and properties. “R-Groups” can be defined inmultiple terms including, but not limited to: individual OTUs,individual or multiple OTUs derived from a specific phylogenetic cladeor a desired phenotype such as the ability to form spores, or functionalbacterial compositions. “Backbone Networks” can comprise acomputationally derived Network Ecology in its entirely or can comprisesubsets of the computed network that represent key nodes in the networkthat contribute to efficacy such as but not limited to a composition ofKeystone OTUs. The number of organisms in a human gastrointestinaltract, as well as the diversity between healthy individuals, isindicative of the functional redundancy of a healthy gut microbiomeecology. See The Human Microbiome Consortia. 2012. Structure, functionand diversity of the healthy human microbiome. Nature 486: 207-214, Thisredundancy makes it highly likely that non-obvious subsets of OTUs orfunctional pathways (i.e. “Backbone Networks”) are critical tomaintaining states of health and/or catalyzing a shift from a dysbioticstate to one of health. One way of exploiting this redundancy is throughthe substitution of OTUs that share a given clade (see below) or byadding members of a clade not found in the Backbone Network.

“Bacterial Composition” refers to a composition comprising bacteria,and/or bacterial spores. In some embodiments, a bacterial compositionincludes a consortium of microbes comprising two or more OTUs, BackboneNetwork Ecologies, Functional Network Ecologies, Network Classes, andCore Ecologies are all types of bacterial compositions. As used herein,Bacterial Composition includes a therapeutic microbial composition, aprophylactic microbial composition, a Spore Population, a Purified SporePopulation, or an ethanol treated spore population.

“Bacterial translocation” refers to the passage of one or more bacteriaacross the epithelial layer of any organ of a human or non-human animal.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree (i.e.tips of the tree) that are a distinct monophyletic evolutionary unit andthat share some extent of sequence similarity. Clades are hierarchical,in one embodiment, the node in a phylogenetic tree that is selected todefine a clade is dependent on the level of resolution suitable for theunderlying data used to compute the tree topology.

The “colonization” of a host organism includes the non-transitoryresidence of a bacterium or other microscopic organism. As used herein,“reducing colonization” of a host subject's gastrointestinal tract orvagina (or any other microbiota niche) by a pathogenic or non-pathogenicbacterium includes a reduction in the residence time of the bacterium inthe gastrointestinal tract or vagina as well as a reduction in thenumber (or concentration) of the bacterium in the gastrointestinal tractor vagina, or adhered to the luminal surface of the gastrointestinaltract. The reduction in colonization can be permanent or occur during atransient period of time. Measuring reductions of adherent pathogens canbe demonstrated directly, e.g., by determining pathogenic burden in abiopsy sample, or reductions may be measured indirectly, e.g., bymeasuring the pathogenic burden in the stool of a mammalian host.

A “Combination” of two or more bacteria includes the physicalco-existence of the two bacteria, either in the same material or productor in physically connected products, as well as the temporalco-administration or co-localization of the two bacteria.

“Cytotoxic” activity of a bacterium includes the ability to kill a cell,e.g., a bacterial cell, such as a pathogenic bacterial cell, or a hostcell. A “cytostatic” activity of a bacterium includes the ability toinhibit (e.g., partially or fully) the growth, metabolism, and/orproliferation of a cell, e.g., a bacterial cell, such as a pathogenicbacterial cell. Cytotoxic activity may also apply to other cell typessuch as but not limited to eukaryotic cells, e.g., host cells.

The term “distal” generally is used in relation to the gastrointestinaltract, specifically the intestinal lumen, of a human or other mammal.Thus, a “distal dysbiosis” includes a dysbiosis outside of the lumen ofthe gastrointestinal tract, and a “distal microbiota” includes amicrobiota outside of the lumen of the gastrointestinal tract. Inspecified instances, the term “distal” may be used in relation to thesite of administration, engraftment, or colonization of a composition,e.g., a probiotic composition, of the invention. For example, if aprobiotic composition is administered vaginally, a “distal” effect ofthe composition would occur outside the vagina.

“Dysbiosis” refers to a state of the microbiota or microbiome of the gutor other body area, including, e.g., mucosal or skin surfaces (or anyother microbiota niche) in which the normal diversity and/or function ofthe ecological network is disrupted. Any disruption from the preferred(e.g., ideal) state of the microbiota can be considered a dysbiosis,even if such dysbiosis does not result in a detectabie decrease inhealth. This state of dysbiosis may be unhealthy (e.g., result in adiseased state), or it may be unhealthy under only certain conditions,or it may prevent a subject from becoming healthier. Dysbiosis may bedue to a decrease in diversity of the microbiota population composition,the overgrowth of one or more population of pathogens (e.g., apopulation of pathogenic bacteria) or pathobionts, the presence ofand/or overgrowth of symbiotic organisms able to cause disease only whencertain genetic and/or environmental conditions are present in apatient, or the shift to an ecological network that no longer provides abeneficial function to the host and therefore no longer promotes health.A “distal dysbiosis” includes, but is not limited to, a dysbiosisoutside of the lumen of the gastrointestinal tract.

“Germinant” is a material or composition, or a physical-chemicalprocess, capable of inducing the germination of vegetative bacterialcells from dormant spores, or the proliferation of vegetative bacterialcells, either directly or indirectly in a host organism and/or in vitro.

“Graft versus host disease” as used herein is an immunological disorderin which the immune cells of a transplant attack the tissues of atransplant recipient, potentially leading to organ dysfunction.

“Acute GVHD” as used herein is GVHD that presents within the first 100days of transplant.

“Chronic GVHD” as used herein is GVHD that presents after the first 100days of transplant.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

“Inhibition” of a pathogen or non-pathogen encompasses the inhibition ofany desired function or activity of the pathogen or non-pathogen by theprobiotic, e.g., bacterial, compositions of the present invention.Demonstrations of inhibition, such as a decrease in the growth of apathogenic bacterial cell population or a reduction in the level ofcolonization of a pathogenic bacterial species are provided herein andotherwise recognized by one of ordinary skill in the art. Inhibition ofa pathogenic or non-pathogenic bacterial population's “growth” mayinclude inhibiting an increase in the size of a pathogenic ornon-pathogenic bacterial cell population and/or inhibiting theproliferation (or multiplication) of a pathogenic or non-pathogenicbacterial cell population. Inhibition of colonization of a pathogenic ornon-pathogenic bacterial species may be demonstrated by measuring andcomparing the amount or burden of the bacterial species before and aftera treatment. An “inhibition” or the act of “inhibiting” includes thetotal cessation and partial reduction of one or more activities of apathogen, such as growth, proliferation, colonization, and function. Asused herein, inhibition includes cytostatic and/or cytotoxic activities.Inhibition of function includes, for example, the inhibition ofexpression of a pathogenic gene product (e.g., the genes encoding atoxin and/or toxin biosynthetic pathway, or the genes encoding astructure required for intracellular invasion (e.g., an invasive pilus))induced by the bacterial composition.

“Isolated” encompasses a bacterium or other entity or substance that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature or in anexperimental setting), and/or (2) produced, prepared, purified, and/ormanufactured by the hand of man. Isolated bacteria includes, forexample, those bacteria that are cultured, even if such cultures are notmonocultures. Isolated bacteria may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated bacteria aremore than about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. The terms “purify,”“purifying” and “purified” refer to a bacterium or other material thathas been separated from at least some of the components with which itwas associated either when initially produced or generated (e.g.,whether in nature or in an experimental setting), or during any timeafter its initial production. A bacterium or a bacterial population maybe considered purified if it is isolated at or after production, such asfrom a material or environment containing the bacterium or bacterialpopulation, or by passage through culture, and a purified bacterium orbacterial population may contain other materials up to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.about 90%. or above about 90% o and still be considered “isolated.” Insome embodiments, purified bacteria and bacterial populations are morethan about 80% o, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%. about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of bacterial compositionsprovided herein, the one or more bacterial types present in thecomposition can be independently purified from one or more otherbacteria produced and/or present in the material or environmentcontaining the bacterial type. In some embodiments, bacterialcompositions and the bacterial components thereof are purified fromresidual habitat products. In other embodiments, bacterial compositionscontain a defined mixture of isolated bacteria. For example, in someembodiments, the probiotic composition contains no more than 100bacterial species. For example, in some embodiments, the probioticcomposition contains no more than 75 bacterial species. In otherembodiments, the probiotic composition contains no more than 50bacterial species, e.g., no more than 40 bacterial species, no more than30 bacterial species, no more than 25 bacterial species, no more than 20bacterial species, no more than 15 bacterial species, no more than 10bacterial species, etc. In other embodiments, the probiotic compositioncontains no more than 10 bacterial species, e.g., 10 bacterial species,9 bacterial species, 8 bacterial species, 7 bacterial species, 6bacterial species, 5 bacterial species, 4 bacterial species, 3 bacterialspecies, 2 bacterial species, 1 bacterial species. In some embodiments,the probiotic composition contains defined quantities of each bacterialspecies. In an exemplary embodiment, the probiotic composition containsisolated bacterial populations that are not isolated from fecal matter.

“Keystone OTU” or “Keystone Function” refers to one or more OTUs orFunctional Pathways (e.g. KEGG or COG pathways) that are common to manynetwork ecologies or functional network ecologies and are members ofnetworks that occur in many subjects (i.e. “are pervasive). Due to theubiquitous nature of Keystone OTUs and their associated FunctionsPathways, they are central to the function of network ecologies inhealthy subjects and are often missing or at reduced levels in subjectswith disease. Keystone OTUs and their associated functions may exist inlow, moderate, or high abundance in subjects. A “non-Keystone OTU” or“non-Keystone Function” refers to an OTU or Function that is observed ina Network Ecology or a Functional Network Ecology and is not a keystoneOTU or Function.

“Metabolism” or “metabolic reaction” as used herein refers to any andall biomolecular catabolic or anabolic processes occurring orpotentially occurring in mammalian cells or in microbes.

“Metabolite” as used herein refers to any and all molecular compounds,compositions, molecules, ions, co-factors, catalysts or nutrients usedas substrates in any cellular or microbial metabolic reaction orresulting as product compounds, compositions, molecules, ions,co-factors, catalysts or nutrients from any cellular or microbialmetabolic reaction.

“Microbiota” refers to the community of microorganisms that inhabit(sustainably or transiently) in and/or on asubject, (e.g, a mammal suchas a human), including, but not limited to, eukaryotes (e.g., protozoa),archaea, bacteria, and viruses (including bacterial viruses, i.e., aphage).

“Microbiome” refers to the genetic content of the communities ofmicrobes that live in and on the human body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)), wherein “genetic content”includes genomic DNA, RNA such as ribosomal RNA, the epigenome,plasmids, and all other types of genetic information.

“Microbial Carriage” or simply “Carriage” refers to the population ofmicrobes inhabiting a niche within or on a subject (e.g., a humansubject). Carriage is often defined in terms of relative abundance. Forexample, OTU1 comprises 60% of the total microbial carriage, meaningthat OTU1 has a relative abundance of 60% compared to the other OTUs inthe sample from which the measurement is made. Carriage is most oftenbased on genomic sequencing data where the relative abundance orcarriage of a single OTU or group of OTUs is defined by the number ofsequencing reads that are assigned to that OTU/s relative to the totalnumber of sequencing reads for the sample.

“Microbial Augmentation” refers to the establishment or significantincrease of a population of microbes that are (i) absent or undetectable(as determined by the use of standard genomic, biochemical and/ormicrobiological techniques) from the administered therapeutic microbialcomposition, and/or (ii) absent, undetectable, or present at lowfrequencies in the host niche (as an example: gastrointestinal tract,skin, anterior-nares, or vagina) before the delivery of the microbialcomposition; and (iii) are found, i.e, detectable, after theadministration of the microbial composition or significantly increase,for instance increase in abundance by 2-fold, 5-fold, 1×10², 1×10³,1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or greater than 1×10⁸, in cases where theyare present at low frequencies. The microbes that comprise an augmentedecology can be derived from exogenous sources such as food and theenvironment, or grow out from micro-niches within the host where theyreside at low frequency.

The administration of the therapeutic composition can induce anenvironmental shift in the target niche that promotes favorableconditions for the growth of commensal microbes. In the absence oftreatment with a therapeutic microbial composition, with or without oneor more prebiotics, the host can be constantly exposed to thesemicrobes; however, sustained growth and the positive health effectsassociated with the stable population of increased levels of themicrobes comprising the augmented ecology are not observed.

“Microbial Engraftment” or simply “engraftment” refers to theestablishment of OTUs comprised in a therapeutic microbial compositionin a target niche. In one embodiment, the OTUs are absent in the treatedhost prior to treatment. The microbes that comprise the engraftedecology are found in the therapeutic microbial composition and establishas constituents of the host microbial ecology upon treatment. EngraftedOTUs can establish for a transient period of time, or demonstratelong-term stability in the microbial ecology that populates the hostpost-treatment with a therapeutic microbial composition. The engraftedecology can induce an environmental shift in the target niche thatpromotes favorable conditions for the growth of commensal microbescapable of catalyzing a shift from a dysbiotic ecology to onerepresentative of a healthy state.

As used herein, the term “minerals” is understood to include boron,calcium, chromium, copper, iodine, iron, magnesium, manganese,molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin,vanadium, zinc, or combinations thereof.

“Network Ecology” refers to a consortium of clades or OTUs that co-occurin some number of subjects. As used herein, a “network” is definedmathematically by a graph delineating how specific nodes (i.e. clades orOTUs) and edges (connections between specific clades or OTUs) relate toone another to define the structural ecology of a consortium of cladesor OTUs. Any given Network Ecology will possess inherent phylogeneticdiversity and functional properties.

A Network Ecology can also be defined in terms of its functionalcapabilities where for example the nodes would be comprised of elementssuch as, but not limited to, enzymes, clusters of orthologous groups(COGS; http://www.ncbi.nlm.nih.gov books/NBK21090/), or KEGG OrthologyPathways (www.genome.jp/kegg/); these networks are referred to as a“Functional Network Ecology”. Functional Network Ecologies can bereduced to practice by defining the group of OTUs that together comprisethe functions defined by the Functional Network Ecology.

The terms “Network Class”, “Core Network” and “Network Class Ecology”refer to a group of network ecologies that in general arecomputationally determined to comprise ecologies with similarphylogenetic and/or functional characteristics. A Network Classtherefore contains important biological features, defined eitherphylogenetically or functionally, of a group (i.e., a cluster) ofrelated network ecologies. One representation of a Core Network Ecologyis a designed consortium of microbes, typically non-pathogenic bacteria,that represents core features of a set of phylogenetically orfunctionally related network ecologies seen in many different subjects.In many occurrences, a Core Network, while designed as described herein,exists as a Network Ecology observed in one or more subjects. CoreNetwork ecologies are useful for reversing or reducing a dysbiosis insubjects where the underlying, related Network Ecology has beendisrupted.

“Ecological Niche” or simply “Niche” refers to the ecological space thatan organism or group of organisms (e.g., a bacterial population)occupies. Niche describes how an organism or population or organismsresponds to the distribution of resources, physical parameters (e.g.,host tissue space) and competitors (e.g., by growing when resources areabundant, and/or when predators, parasites and pathogens are scarce) andhow it in turn alters those same factors (e.g., limiting access toresources by other organisms, acting as a food source for predators anda consumer of prey).

To be free of “non-comestible products” means that a bacterialcomposition or other material provided herein does not have asubstantial amount of a non-comestible product, e.g., a product ormaterial that is inedible, harmful or otherwise undesired in a productsuitable for administration, e.g., oral administration, to a humansubject.

“Operational taxonomic units,” “OTU” (or plural, “OTUs”) refer to aterminal leaf in a phylogenetic tree and is defined by a nucleic acidsequence, e.g., the entire genome, or a specific genetic sequence, andall sequences that share sequence identity to this nucleic acid sequenceat the level of species. In some embodiments the specific geneticsequence may be the 16S sequence or a portion of the 16S sequence. Inother embodiments, the entire genomes of two entities are sequenced andcompared. In another embodiment, select regions such as multilocussequence tags (MLST), specific genes, or sets of genes may begenetically compared. In 16S embodiments, OTUs that share ≧97% averagenucleotide identity across the entire 16S or some variable region of the16S are considered the same OTU (see e.g. Claesson M J, Wang Q,O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010.Comparison of two next-generation sequencing technologies for resolvinghighly complex microbiota composition using tandem variable 16S rRNAgene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A,and Tiedje J M. 2006. The bacterial species definition in the genomicera. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). In embodimentsinvolving the complete genome, MLSTs, specific genes, or sets of genesOTUs that share ≧95% average nucleotide identity are considered the sameOTU (see e.g. Achtman M, and Wagner M. 2008. Microbial diversity and thegenetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940.). OTUs are frequently defined by comparing sequencesbetween organisms. Generally, sequences with less than 95% sequenceidentity are not considered to form part of the same OTU. OTUs may alsobe characterized by any combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof. Such characterization employs, e.g., WGS data or awhole genome sequence.

“Pathobionts” or “Opportunistic Pathogens” refers to symbiotic organismsable to cause disease only when certain genetic and/or environmentalconditions are present in a subject.

The term “Phylogenetic Diversity” refers to the biodiversity present ina given Network Ecology, Core Network Ecology or Network Class Ecologybased on the OTUs that comprise the network. Phylogenetic diversity is arelative term, meaning that a Network Ecology, Core Network or NetworkClass that is comparatively more phylogenetically diverse than anothernetwork contains a greater number of unique species, genera, andtaxonomic families. Uniqueness of a species, genera, or taxonomic familyis generally defined using a phylogenetic tree that represents thegenetic diversity all species, genera, or taxonomic families relative toone another. In another embodiment phylogenetic diversity may bemeasured using the total branch length or average branch length of aphylogenetic tree.

Phylogenetic Diversity may be optimized in a bacterial composition byincluding a wide range of biodiversity.

“Phylogenetic tree” refers to a graphical representation of theevolutionary relationships of one genetic sequence to another that isgenerated using a defined set of phylogenetic reconstruction algorithms(e.g. parsimony, maximum likelihood, or Bayesian). Nodes in the treerepresent distinct ancestral sequences and the confidence of any node isprovided by a bootstrap or Bayesian posterior probability, whichmeasures branch uncertainty.

As used herein “preventing” or “prevention” refers to any methodologywhere the disease state does not occur due to the actions of themethodology (such as, for example, administration of a probiotic and/ora prebiotic as described herein). In one aspect, it is understood thatprevention can also mean that the disease is not established to theextent that occurs in untreated controls. For example, there can be a 5,10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100% reduction in theestablishment of disease frequency relative to untreated controls.Accordingly, prevention of a disease encompasses a reduction in thelikelihood that a subject will develop the disease, relative to anuntreated subject (e.g. a subjece who does not receive a probioticand/or a prebiotic as described herein).

“rDNA”, “rRNA”, “16S-rDNA”, “16S-rRNA”, “16S”, “16S sequencing”,“16S-NGS”, “18S”, “18S-rRNA”, “18S-rDNA”, “18S sequencing”, and“18S-NGS” refer to the nucleic acids that encode for the RNA subunits ofthe ribosome. rDNA refers to the gene that encodes the rRNA thatcomprises the RNA subunits. There are two RNA subunits in the ribosometermed the small subunit (SSU) and large subunit (LSU); the RNA geneticsequences (rRNA) of these subunits are related to the gene that encodesthem (rDNA) by the genetic code. rDNA genes and their complementary RNAsequences are widely used for determination of the evolutionaryrelationships amount organisms as they are variable, yet sufficientlyconserved to allow cross organism molecular comparisons.

Typically 16S rDNA sequence (approximately 1542 nucleotides in length)of the 30S SSU is used for molecular-based taxonomic assignments ofProkaryotes and the 18S rDNA sequence (approximately 1869 nucleotides inlength) of 40S SSU is used for Eukaryotes. 16S sequences are used forphylogenetic reconstruction as they are in general highly conserved, butcontain specific hypervariable regions that harbor sufficient nucleotidediversity to differentiate genera and species of most bacteria.

“Residual habitat products” refers to material derived from the habitatfor microbiota within or on a human or animal. For example, microbiotalive in feces in the gastrointestinal tract, on the skin itself, insaliva, mucus of the respiratory tract, or secretions of thegenitourinary tract (i.e., biological matter associated with themicrobial community). Substantially free of residual habitat productsmeans that the bacterial composition no longer contains the biologicalmatter associated with the microbial environment on or in the human oranimal subject and is 100% free, 99% free, 98% free, 97% free, 96% free,or 95% free, 94% free, 93% free, 92% free, 91% free, 90% free, 85% free,80% free, 75% free, 70% free, 65% free, or 60% free of any contaminatingbiological matter associated with the microbial community. Residualhabitat products can include abiotic materials (including undigestedfood) or it can include unwanted microorganisms. Substantially free ofresidual habitat products may also mean that the bacterial compositioncontains no detectable cells from a human or animal and that onlymicrobial cells are detectable. In one embodiment, substantially free ofresidual habitat products may also mean that the bacterial compositioncontains no detectable viral (including bacterial viruses (i.e.,phage)), fungal, mycoplasmal contaminants. In another embodiment, itmeans that fewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%,1×10⁻⁷%, 1×10⁻⁸% of the viable cells in the bacterial composition arehuman or animal, as compared to microbial cells. There are multiple waysto accomplish this degree of purity, none of which are limiting. Thus,contamination may be reduced by isolating desired constituents throughmultiple steps of streaking to single colonies on solid media untilreplicate (such as, but not limited to, two) streaks from serial singlecolonies have shown only a single colony morphology. Alternatively,reduction of contamination can be accomplished by multiple rounds ofserial dilutions to single desired cells (e.g., a dilution of 10⁻⁸ or10⁻⁹), such as through multiple 10-fold serial dilutions. This canfurther be confirmed by showing that multiple isolated colonies havesimilar cell shapes and Gram staining behavior. Other methods forconfirming adequate purity include genetic analysis (e.g. PCR, DNAsequencing), serology and antigen analysis, enzymatic and metabolicanalysis, and methods using instrumentation such as flow cytometry withreagents that distinguish desired constituents from contaminants.

In microbiology, “16S sequencing” or “16S-rRNA” or “16S” refers tosequence derived by characterizing the nucleotides that comprise the 16Sribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500nucleotides in length and is used in reconstructing the evolutionaryrelationships and sequence similarity of one bacterial isolate toanother using phylogenetic approaches. 16S sequences are used forphylogenetic reconstruction as they are in general highly conserved, butcontain specific hypervariable regions that harbor sufficient nucleotidediversity to differentiate genera and species of most bacteria.

The “V1-V9 regions” of the 16S rRNA refers to the first through ninthhypervariable regions of the 16S rRNA gene that are used for genetictyping of bacterial samples. These regions in bacteria are defined bynucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043,1117-1173, 1243-1294 and 1435-1465 respectively using numbering based onthe E. coli system of nomenclature. Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS75(10):4801-4805 (1978). In some embodiments, at least one of the V1,V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize anOTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. A person of ordinary skill in the art canidentify the specific hypervariable regions of a candidate 16S rRNA bycomparing the candidate sequence in question to a reference sequence andidentifying the hypervariable regions based on similarity to thereference hypervariable regions, or alternatively, one can employ WholeGenome Shotgun (WGS) sequence characterization of microbes or amicrobial community.

The term “subject” refers to any organism or animal subject that is anobject of a method or material, including mammals, e.g., humans,laboratory animals (e.g., primates, rats, mice, rabbits), livestock(e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets(e.g., dogs, cats, and rodents), horses, and transgenic non-humananimals. The subject may be suffering from a dysbiosis, including, butnot limited to, an infection due to a gastrointestinal pathogen or maybe at risk of developing or transmitting to others an infection due to agastrointestinal pathogen. Synonyms used herein include “patient” and“animal.” In some embodiments, the subject or host may be suffering froma dysbiosis, that contributes to or causes a condition classified as anautoimmune or inflammatory disease, graft-versus-host disease, Crohn'sdisease, Celiac disease, inflammatory bowel disease, ulcerative colitis,multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, ortype 1 diabetes. In some embodiments, the host may be suffering fromincluding but not limited to mechanisms such as metabolic endotoxemia,altered metabolism of primary bile acids, immune system activation, oran imbalance or reduced production of short chain fatty acids includingbutyrate, propionate, acetate, and branched chain fatty acids.

The term “phenotype” refers to a set of observable characteristics of anindividual entity. As example an individual subject may have a phenotypeof “health” or “disease”. Phenotypes describe the state of an entity andall entities within a phenotype share the same set of characteristicsthat describe the phenotype. The phenotype of an individual results inpart, or in whole, from the interaction of the entities genome and/ormicrobiome with the environment.

“Spore” or “endospore” refers to an entity, particularly a bacterialentity, which is in a dormant, non-vegetative and non-reproductivestage. Spores are generally resistant to environmental stress such asradiation, desiccation, enzymatic treatment, temperature variation,nutrient deprivation, and chemical disinfectants.

A “spore population” refers to a plurality of spores present in acomposition. Synonymous terms used herein include spore composition,spore preparation, ethanol treated spore fraction and spore ecology. Aspore population may be purified from a fecal donation, e.g. via ethanolor heat treatment, or a density gradient separation or any combinationof methods described herein to increase the purity, potency and/orconcentration of spores in a sample. Alternatively, a spore populationmay be derived through culture methods starting from isolated sporeformer species or spore former OTUs or from a mixture of such species,either in vegetative or spore form.

A “sporulation induction agent” is a material or physical-chemicalprocess that is capable of inducing sporulation in a bacterium, eitherdirectly or indirectly, in a host organism and/or in vitro.

To increase production of bacterial entities includes an activity or asporulation induction agent. Production includes conversion ofvegetative bacterial cells into spores and augmentation of the rate ofsuch conversion, as well as decreasing the germination of bacteria inspore form, decreasing the rate of spore decay in vivo, or ex vivo, orto increasing the total output of spores (e.g. via an increase involumetric output of fecal material).

“Synergy” or “synergistic interactions” refers to the interaction orcooperation of two or more microbes to produce a combined effect greaterthan the sum of their separate effects. In one embodiment, “synergy”between two or more microbes can result in the inhibition of a pathogensability to grow.

“Treatment,” “treat,” or “treating” means a method of reducing theeffects of a disease or condition. Treatment can also refer to a methodof reducing the disease or condition itself rather than just thesymptoms. The treatment can be any reduction from pre-treatment levelsand can be but is not limited to the complete ablation of the disease,condition, or the symptoms of the disease or condition. Therefore, inthe disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease or the disease progression. For example, a disclosedmethod for reducing the effects of GVHD is considered to be a treatmentif there is a 10% reduction in one or more symptoms of the disease in asubject with GVHD when compared to pre-treatment levels in the samesubject or control subjects. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels. It is understood and hereincontemplated that “treatment” does not necessarily refer to a cure ofthe disease or condition, but an improvement in the outlook of a diseaseor condition (e.g., GVHD).

As used herein the term “vitamin” is understood to include any ofvarious fat-soluble or water-soluble organic substances (non-limitingexamples include vitamin A, Vitamin B1 (thiamine), Vitamin B2(riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5(pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine,or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folicacid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin invitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 andK2 (i.e. MK-4, MK-7), folic acid and biotin) essential in minute amountsfor normal growth and activity of the body and obtained naturally fromplant and animal foods or synthetically made, pro-vitamins, derivatives,analogs. As used herein, the term “recipient” refers to the subjectreceives a bone marrow or a solid organ transplantation.

III. Probiotic Compositions of the Invention

Disclosed herein are bacterial, e.g., probiotic, compositions comprisinga non-pathogenic bacterial or fungal population, e.g., animmunomodulatory bacterial population, such as an anti-inflammatorybacterial population, with or without one or more prebiotics, for theprevention, control, and treatment of transplant disorders, and forgeneral nutritional health in a subject receiving a transplant. Thesecompositions are advantageous in being suitable for safe administrationto humans and other mammalian subjects and are efficacious for thetreatment, prevention, reduction and amelioration of graft versus hostdisease (GVHD), and complications associated therewith, such astransplant rejection. While spore-based compositions are known, theseare generally prepared according to various techniques such aslyophilization or spray-drying of liquid bacterial cultures, resultingin poor efficacy, instability, substantial variability and lack ofadequate safety and efficacy.

It has now been found that bacterial and fungal populations can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and administered to mammalian subjects using themethods as provided herein.

As described in detail herein, alterations in the microbiota of atransplant recipient significantly impact the outcome for the subject.In particular, a dysbiosis in the gastrointestinal tract, or a dysbiosisdistal to the gastrointestinal tract, can increase the likelihood that asubject will develop GVHD, and reduce the overall survival of thesubject following the transplant. Outcome can be improved byadministering a probiotic composition, optionally in combination with aprebiotic, to correct the dysbiosis. In particular, probioticcompositions that improve intestinal barrier integrity and/or reduceinflammation in the subject can treat or prevent GVHD in a subjectreceiving a transplant.

In one embodiment, therapeutic compositions are provided for thetreatment, prevention, reduction of onset and amelioration ofinflammation or one or more symptom of a transplant disorder, such as,for example, GVHD. As used herein, “therapeutic” compositions includecompositions that function in a prophylactic (e.g., preventative)manner. Therapeutic compositions can contain one or more populations ofimmunomodulatory bacteria and/or fungi, alone or in combination with oneor more prebiotics. In one embodiment, the microbial entities can beproduced by isolation and/or culture, using, for example, the followingsteps: a) providing fecal material and b) subjecting the material to aculture step and/or a treatment step resulting in purification and/orisolation of immunomodulatory bacteria and, optionally, c) formulatingthe purified population for administration, wherein the purifiedpopulation is present in the composition in an amount effective toengraft and/or colonize in the gastrointestinal tract in order to treat,prevent or reduce the severity of inflammation or one or more symptom ofGVHD in a mammalian recipient subject to whom the therapeuticcomposition is administered. Generally, the population is provided in anamount effective to treat (including to prevent) a disease, disorder orcondition associated with or characterized by inflammation or dysbiosis,e.g., transplant rejection or GVHD. Such treatment may be effective toreduce the severity of at least one symptom of the dysbiosis, e.g.,gastrointestinal or distal dysbiosis, thereby improving survival of thetransplant recipient. Such treatment may be effective to modulate themicrobiota diversity present in the mammalian recipient.

In embodiments, the probiotic compositions contain immunomodulatorymicrobes, e.g., immunomodulatory bacteria, which are capable of alteringthe immune activity of a mammalian subject. In exemplary embodiments,the immunomodulatory bacteria are capable of reducing inflammation in amammalian subject. Such immunomodulatory bacteria are referred to hereinas anti-inflammatory bacteria. Immunomodulatory bacteria can act toalter the immune activity of a subject directly or indirectly. Forexample, immunomodulatory bacteria can act directly on immune cellsthrough receptors for bacterial components (e.g. Toll-like receptors) orby producing metabolites such as immunomodulatory short chain fattyacids (SCFAs). SCFAs produced by immunomodulatory bacteria can include,e.g., butyrate, acetate, propionate, or valerate, or combinationsthereof. Such SCFAs can have many positive impacts on the health of thesubject, by, for example, reducing inflammation, or improving intestinalbarrier integrity. In one embodiment, the improvement of gut epitheliumbarrier integrity results in reduced trafficking of bacteria, bacterialcomponents and/or bacterial metabolites into the blood. In oneembodiment, a probiotic composition is administered to a subject in anamount effective to increase short chain fatty acid production by one ormore organisms in the gut of a mammalian host. Immunomodulatory bacteriacan also impact the immune activity of a subject by producingglutathione or gamma-glutamylcysteine. Probiotics containing suchimmunomodulatory bacteria can treat or prevent GVHD in a subjectreceiving a transplant.

Probiotic compositions containing immunomodulatory bacteria canadditionally or alternatively impact the immune activity of a subjectindirectly by modulating the activity of immune cells in the subject.For example, immunomodulatory bacteria may alter cytokine expression byhost immune cells (e.g., macrophages, B lymphocytes, T lymphocytes, mastcells, peripherial blood mononuclear cells (PBMCs), etc.) or other typesof host cells capable of cytokine secretion (e.g., endothelia cells,fibroblasts, stromal cells, etc.). In an exemplary embodiment, probioticcompositions contain anti-inflammatory immunomodulatory bacteria thatare capable of inducing secretion of anti-inflammatory cytokines by hostcells. For example, anti-inflammatory bacteria can induce secretion ofone or more anti-inflammatory cytokines such as but not limited toIL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, and combinations thereof, by hostcells (e.g., host immune cells). In another exemplary embodiment,probiotic compositions contain anti-inflammatory immunomodulatorybacteria that are capable of reducing secretion of one or morepro-inflammatory cytokines by host cells (e.g., host immune cells). Forexample, anti-inflammatory bacteria can reduce secretion of one or morepro-inflammatory cytokines such as but not limited to IFNγ, IL-12p70,IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof.Other cytokines that may be modulated by immunomodulatory bacteriainclude, for example, IL-17A, IL-2, and IL-9. In some embodiments, theinduction and/or secretion of pro-inflammatory cytokines may be inducedby (e.g., in response to, either directly or indirectly) a bacteria(e.g., Enterococcus faecalis).

In some embodiments, immunomodulatory bacteria are selected forinclusion in a probiotic composition of the invention based on thedesired effect of the probiotic composition on cytokine secretion byhost cells, e.g., host immune cells. For example, in one embodiment, aprobiotic composition contains anti-inflammatory bacteria that increasesecretion of an anti-inflammatory cytokine, for example, IL-10, IL-13,IL-9, IL-4, IL-5, TGFβ, and combinations thereof. In some embodiments,the anti-inflammatory bacteria increase secretion of two or moreanti-inflammatory cytokines. In some embodiments, the anti-inflammatorybacteria increase secretion of three or more anti-inflammatorycytokines. In some embodiments, the anti-inflammatory bacteria increasesecretion of four or more anti-inflammatory cytokines. In someembodiments, the anti-inflammatory bacteria increase secretion of fiveor more anti-inflammatory cytokines. In exemplary embodiments, theincrease is an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 80%, 100%, 200%, 300%, 500% or more. In another embodiment, aprobiotic composition contains anti-inflammatory bacteria that decreasesecretion of a pro-inflammatory cytokine, for example, IFNγ, IL-12p70,IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof.In some embodiments, the anti-inflammatory bacteria decrease secretionof two or more pro-inflammatory cytokines. In some embodiments, theanti-inflammatory bacteria decrease secretion of three or morepro-inflammatory cytokines. In some embodiments, the anti-inflammatorybacteria decrease secretion of four or more pro-inflammatory cytokines.In some embodiments, the anti-inflammatory bacteria decrease secretionof five or more pro-inflammatory cytokines. In exemplary embodiments,the decrease is a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 80%, 100%, 200%, 300%, 500% or more. In another embodiment,the probiotic composition contains anti-inflammatory bacteria thatincrease secretion of one or more anti-inflammatory cytokines and reducesecretion of one or more pro-inflammatory cytokines. Alterations incytokine expression may occur locally, e.g., in the gastrointestinaltract of a subject, or at a site distal to the gastrointestinal tract.Such anti-inflammatory bacteria may be used to treat or prevent GVHD ina transplant recipient.

In other embodiments, probiotics containing immunomodulatory bacteriaimpact the immune activity of a subject by promoting the differentiationand/or expansion of particular subpopulations of immune cells. Forexample, immunomodulatory bacteria can increase or decrease theproportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in asubject. The increase or decrease in the proportion of immune cellsubpopulations may be systemic, or it may be localized to a site ofaction of the probiotic, e.g., in the gastrointestinal tract or at thesite of a distal dysbiosis. In some embodiments, immunomodulatorybacteria are selected for inclusion in a probiotic composition of theinvention based on the desired effect of the probiotic composition onthe differentiation and/or expansion of subpopulations of immune cellsin the subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria that increase the proportion of Treg cells in a subject. Inanother embodiment, a probiotic composition contains immunomodulatorybacteria that decrease the proportion of Treg cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th17 cells in a subject (e.g., byinducing expansion of Th17 cells in the subject). In another embodiment,a probiotic composition contains immunomodulatory bacteria that decreasethe proportion of Th17 cells in a subject. In one embodiment, aprobiotic composition contains immunomodulatory bacteria that increasethe proportion of Th1 cells in a subject (e.g., by inducing expansion ofTh1 cells in the subject). In another embodiment, a probioticcomposition contains immunomodulatory bacteria that decrease theproportion of Th1 cells in a subject. In one embodiment, a probioticcomposition contains immunomodulatory bacteria that increase theproportion of Th2 cells in a subject (e.g., by inducing expansion of Th2cells in the subject). In another embodiment, a probiotic compositioncontains immunomodulatory bacteria that decrease the proportion of Th2cells in a subject. The increase or decrease in the proportion of immunecell subpopulations (e.g., Th17 cells, Th1 cells and Th2 cells) may belocalized or systemic.

In one embodiment, a probiotic composition contains immunomodulatorybacteria capable of modulating the proportion of one or more populationsof Treg cells, Th17 cells, Th1 cells, Th2 cells, and combinationsthereof in a subject. Certain immune cell profiles may be particularlydesirable to treat or prevent particular disorders associated with adysbiosis. For example, treatment or prevention of GVHD can be promotedby increased numbers of Treg cells and Th2 cells, and/or decreasednumbers of Th17 cells and Th1 cells. Accordingly, probiotic compositionsfor the treatment or prevention of GVHD may contain probiotics capableof promoting Treg cells and Th2 cells, and reducing Th17 and Th1 cells.

In one embodiment, therapeutic probiotic compositions comprising apurified population of immunomodulatory microbes, e.g., bacteria, areprovided, with or without one or more prebiotics, in an amount effectiveto i) treat or prevent dysbiosis, e.g., gastrointestinal or distaldysbiosis, inflammation, or an autoimmune or inflammatory disorder,and/or ii) augment at least one type of microbe, e.g., a bacterium, notpresent in the therapeutic composition in a mammalian recipient subjectto whom the therapeutic composition is administered, and/or iii) engraftat least one type of microbe, e.g., a bacterium, present in thetherapeutic composition but not present in a mammalian subject prior totreatment.

In another embodiment, therapeutic probiotic compositions comprising apurified population of immunomodulatory microbes are provided, in anamount effective to i) augment the microbiota diversity present in themammalian recipient and/or ii) treat or prevent dysbiosis, e.g.,gastrointestinal or distal dysbiosis, inflammation, or an autoimmune orinflammatory disorder in a mammalian recipient subject to whom thetherapeutic composition is administered, wherein the purified populationis obtained by separation of the population apart from at least oneresidual habitat product in a fecal material obtained from one or aplurality of mammalian donor subjects. In some embodiments, individualbacterial strains can be cultured from fecal material. These strains canthen be purified or otherwise isolated and used singly or incombination. In one embodiment, the probiotic composition does notcontain a fecal extract.

In one embodiment, the probiotic compositions described herein may beused to treat or correct a dysbiosis in a subject. The dysbiosis may be,for example, a local dysbiosis, or a distal dysbiosis. In anotherembodiment, the probiotic compositions described herein may be used toprevent a dysbiosis in a subject at risk for developing a dysbiosis.

In some embodiments, the purified population of immunomodulatorymicrobes described above is coadministered or coformulated with one ormore prebiotics, e.g., carbohydrates.

In some embodiments, the purified population of immunomodulatorymicrobes described above is administered before one or more prebioticsare administered to a subject. In some embodiments the purifiedpopulation of immunomodulatory microbes is administered after one ormore prebiotics have been administered to a subject. In someembodiments, the purified population of immunomodulatory microbes isadministered concurrently with one or more prebiotics. In otherembodiments, the purified population of immunomodulatory microbes isadministered sequentially with one or more prebiotics. In someembodiments, the purified population of immunomodulatory microbes isadministered in a composition formulated to contain one or morepharmaceutical excipients, and optionally one or more prebiotics.

Microbes involved in modulation of the host immune system i) may behuman commensals; ii) may be part of an organ's healthy-statemicrobiome; ii) may be part of a distal organ's healthy-statemicrobiome; iv) may be exogenous microbes; v) may be innocuous; vi) maybe pathobionts; vii) may be pathogens; viii) may be opportunisticpathogens; or ix) any combination thereof. In some aspects, microbes arenot required to be actively proliferating (e.g., spores, dormant cells,cells with reduced metabolic rate, or heat-killed cells) to have animmunomodulatory effect. In certain aspects, microbial cell components,rather than whole microbial cells, may have immunomodulatory effects.Non-limiting examples of microbial components are lipids, carbohydrates,proteins, nucleic acids, and small molecules.

Microbial compositions are provided herein, optionally comprisingprebiotics, non-microbial immunomodulatory carbohydrates, or microbialimmunomodulatory cell components, that are effective for the preventionor treatment of an autoimmune or inflammatory disorder such asgraft-versus-host disease (GVHD), and/or a dysbiosis which contributesto GVHD.

In certain embodiments, the compositions comprise at least one type ofmicrobe and at least one type of carbohydrate (a prebiotic), andoptionally further comprise microbial immunomodulatory cell componentsor substrates for the production of immunomodulatory metabolites, thatare effective for the prevention or treatment of an autoimmune orinflammatory disorder such as graft-versus-host disease (GVHD). Methodsfor the prevention and/or treatment of GVHD in human subjects are alsodisclosed herein.

In some embodiments, the bacterial, e.g., probiotic, compositions of theinvention comprise purified spore populations. As described herein,purified spore populations contain commensal bacteria of the human gutmicrobiota with the capacity to meaningfully provide one or morefunctions of a healthy microbiota when administered to a mammaliansubject. Without being limited to a specific mechanism, it is thoughtthat such compositions inhibit the growth of pathogens such as C.difficile, Salmonella spp., enteropathogenic E. coli, Fusobacteriumspp., Klebsiella spp. and vancomycin-resistant Enterococcus spp., sothat a healthy, diverse and protective microbiota can be maintained or,in the case of pathogenic bacterial infections, repopulate theintestinal lumen to reestablish ecological control over potentialpathogens. In some embodiments, yeast spores and other fungal spores arealso purified and selected for therapeutic use.

In one embodiment, the purified spore populations can engraft in thehost and remain present for 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 10 days, 14 days, 21 days, 25 days, 30 days, 60 days, 90days, or longer than 90 days. Additionally, the purified sporepopulations can induce other healthy commensal bacteria found in ahealthy gut to engraft in the host that are not present in the purifiedspore populations or present at lesser levels. Therefore, these speciesare considered to “augment” the delivered spore populations. In thismanner, commensal species augmentation of the purified spore populationin the recipient's gut leads to a more diverse population of gutmicrobiota than present initially.

In some embodiments, a probiotic composition of the invention contains asingle species of bacteria. In other embodiments, the probioticcomposition contains two or more species of bacteria, e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 ormore species of bacteria. In one embodiment, the probiotic compositioncontains no more than 20 species of bacteria, e.g., 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 species ofbacteria. In exemplary embodiments, the probiotic composition contains 8bacterial species. In other exemplary embodiments, the probioticcomposition contains 9 bacterial species. In other embodiments, theprobiotic composition contains or is administered in conjunction with aprebiotic, as described herein.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter.

Preferred bacterial genera also include Acetonema, Alkaliphilus,Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor,Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella,Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Halobacteroides, Heliobacterium, Heliophilum, Heliorestis,Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.),Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora,Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum,Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus,Thermoanaerobacter, and Thermosinus.

In another embodiment, a probiotic composition of the invention consistsessentially of Blautia.

In one embodiment, a probiotic composition of the invention does notcomprise Blautia alone.

As provided herein, therapeutic compositions comprise, or in thealternative, modulate, the colonization and/or engraftment, of thefollowing exemplary bacterial entities: Lactobacillus gasseri,Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis,Enterococcus durans, Enterococcus villorum, Lactobacillus plantarum,Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcuscohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcusmitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcusanginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridiumsaccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6,Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcusgnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautiaproducta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonellaparvula, Clostridium methylpentosum, Clostridium islandicum,Faecalibacterium prausnitzii, Bacteroides uniformmis, Bacteroidesthetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus,Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirumpropionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria,Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta.

Preferred bacterial species are provided in Table 1, Table 1A, Table 1B,Table 1C, Table 1D, Table 1E, Table 1F, and Table 5. Optionally, in someembodiments, preferred bacterial species are spore formers. Wherespecific strains of a species are provided, one of skill in the art willrecognize that other strains of the species can be substituted for thenamed strain.

In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Acidaminococcusintestine. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isAcinetobacter baumannii. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Acinetobacter lwoffii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Akkermansia muciniphila. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Alistipes putredinis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Alistipes shahii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerostipes hadrus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerotruncus colihominis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides caccae.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidescellulosilyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isBacteroides dorei. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides eggerthii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides finegoldii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides fragilis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroides massiliensis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides ovatus.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidessalanitronis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBacteroides salyersiae. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides sp. 1_1_6. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides sp. 3_1_23. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides sp. D20. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroidesthetaiotaomicrond. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides uniformis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides vulgatus. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bifidobacterium adolescentis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacterium bifidum. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Bifidobacterium breve.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bifidobacteriumfaecale. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBifidobacterium kashiwanohense. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Bifidobacterium longum subsp. Longum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bifidobacterium stercoris. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Blautia (Ruminococcus) coccoides. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia faecis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia glucerasea. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)hansenii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)luti. In one embodiment, the bacterial entity, e.g., species or strain,useful in the compositions and methods of the invention is Blautia(Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia producta (Ruminococcus productus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)schinkii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia stercoris. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1).In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Blautia unculturedbacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia wexlerae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Candidatus Arthromitus sp.SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isCatenibacterium mitsuokai. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridiales bacterium1_7_47FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium asparagiforme. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridium bolteae. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium clostridioforme. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium glycyrrhizinilyticum. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium (Hungatella)hathewayi. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium histolyticum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium indolis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium leptum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium (Tyzzerella) nexile. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium perfringens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium(Erysipelatoclostridium) ramosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium scindens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium septum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium sp. 14774. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium sp.7_3_54FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium sp. HGF2. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium symbiosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Collinsella aerofaciens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Collinsella intestinalis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Coprobacillus sp. D7. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus catus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus comes. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Dorea formicigenerans.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Dorea longicatena.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Enterococcusfaecalis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEnterococcus faecium. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli S88. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacterium eligens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacteriumfissicatena. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEubacterium ramulus. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Eubacterium rectale. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Faecalibacterium prausnitzii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Flavonifractor plautii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Fusobacterium mortiferum.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Fusobacteriumnucleatum. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isHoldemania filiformis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Hydrogenoanaerobacterium saccharovorans. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Klebsiella oxytoca. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isLachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Lachnospiraceae bacterium 5_1_57FAA. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus casei. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus rhamnosus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Lactobacillus ruminis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Lactococcus casei.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Odoribactersplanchnicus. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isOscillibacter valericigenes. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Parabacteroides gordonii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Parabacteroides johnsonii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Parabacteroides merdae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Pediococcus acidilactici.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Peptostreptococcusasaccharolyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isPropionibacterium granulosum. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Roseburia intestinalis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Roseburia inulinivorans. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Ruminococcus faecis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus gnavus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus sp. ID8. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Ruminococcus torques.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Slackia piriformis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Staphylococcusepidermidis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStaphylococcus saprophyticus. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Streptococcus cristatus. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus dysgalactiae subsp. Equisimilis. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Streptococcus infantis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Streptococcusoralis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStreptococcus sanguinis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Streptococcus viridans. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus thermophiles. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Veillonella dispar.

In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Acidaminococcus intestine. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter baumannii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter lwoffii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Akkermansia muciniphila. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes putredinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes shahii. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Anaerostipes hadrus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Anaerotruncus colihominis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Bacteroides caccae. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides cellulosilyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides dorei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides eggerthii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides finegoldii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides fragilis. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Bacteroidesmassiliensis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Bacteroides ovatus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bacteroides salanitronis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides salyersiae. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 1_1_6. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 3_1_23. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. D20. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides thetaiotaomicrond. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides uniformis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides vulgatus. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium adolescentis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium bifidum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium breve. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium faecale. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium kashiwanohense. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium longum subsp. Longum.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Bifidobacteriumstercoris. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) coccoides. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia faecis. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiaglucerasea. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) hansenii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) luti. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) obeum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia producta (Ruminococcusproductus). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) schinkii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia stercoris. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiauncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia uncultured bacterium cloneSJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia wexlerae. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Catenibacterium mitsuokai. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridiaceae bacterium (Dielmafastidiosa) JC13. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Clostridialesbacterium 1_7_47FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumasparagiforme. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium bolteae.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium clostridioforme. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium glycyrrhizinilyticum. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Hungatella) hathewayi.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium histolyticum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium indolis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium leptum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Tyzzerella) nexile. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium perfringens. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Erysipelatoclostridium)ramosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumscindens. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium septum. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumsp. 14774. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.7_3_54FAA. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.HGF2. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumsymbiosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaaerofaciens. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaintestinalis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprobacillus sp.D7. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprococcus catus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Coprococcus comes. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea formicigenerans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea longicatena. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecalis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecium. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Erysipelotrichaceae bacterium 3_1_53. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Escherichia coli. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Escherichia coli S88. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium eligens. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium fissicatena. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium ramulus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium rectale. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Faecalibacterium prausnitzii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Flavonifractor plautii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium mortiferum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium nucleatum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Holdemania filiformis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Hydrogenoanaerobacterium saccharovorans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Klebsiella oxytoca. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Lachnospiraceaebacterium 7_1_58FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprisesLachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus casei. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus rhamnosus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus ruminis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactococcus casei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOdoribacter splanchnicus. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOscillibacter valericigenes. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides gordonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides johnsonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides merdae. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPediococcus acidilactici. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPeptostreptococcus asaccharolyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Propionibacterium granulosum. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia intestinalis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia inulinivorans. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus faecis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus gnavus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus sp. ID8. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus torques. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Slackia piriformis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus epidermidis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus saprophyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus cristatus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus dysgalactiae subsp. Equisimilis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus infantis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus oralis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus sanguinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus viridans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus thermophiles. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Veillonella dispar.

In some embodiments, the therapeutic composition comprises engineeredmicrobes. For example, engineered microbes include microbes harboring i)one or more genetic changes, such change being an insertion, deletion,translocation, or substitution, or any combination thereof, of one ormore nucleotides contained on the bacterial chromosome or on anendogenous plasmid, wherein the genetic change may result in thealteration, disruption, removal, or addition of one or more proteincoding genes, non-protein-coding genes, gene regulatory regions, or anycombination thereof, and wherein such change may be a fusion of two ormore separate genomic regions or may be synthetically derived; ii) oneor more foreign plasmids containing a mutant copy of an endogenous gene,such mutation being an insertion, deletion, or substitution, or anycombination thereof, of one or more nucleotides; and iii) one or moreforeign plasmids containing a mutant or non-mutant exogenous gene or afusion of two or more endogenous, exogenous, or mixed genes. Theengineered microbe(s) may be produced using techniques including but notlimited to site-directed mutagenesis, transposon mutagenesis,knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemicalmutagenesis, ultraviolet light mutagenesis, transformation (chemicallyor by electroporation), phage transduction, or any combination thereof.Suitable microbes for engineering are known in the art. For example, asdescribed in PCT Publications Nos. WO/93/18163, DELIVERY AND EXPRESSIONOF A HYBRID SURFACE PROTEIN ON THE SURFACE OF GRAM POSITIVE BACTERIA;WO/03/06593, METHODS FOR TREATING CANCER BY ADMINISTERINGTUMOR-TARGETTED BACTERIA AND AN IMMUNOMODULATORY AGENT; andWO/2010/141143, ENGINEERED AVIRULENT BACTERIA STRAINS AND USE IN MEDICALTREATMENTS.

In some embodiments, the engineered microbes are natural humancommensals. In other embodiments, the engineered microbes are attenuatedstrains of pathogens, and may include, but are not limited to,Pseudomonas aeruginosa, Salmonella species, Listeria monocytogenes,Mycoplasma hominis, Escherichia coli, Shigella species, andStreptococcus species, see, e.g. PCT Publications No. WO/03/06593,METHODS FOR TREATING CANCER BY ADMINISTERING TUMOR-TARGETTED BACTERIAAND AN IMMUNOMODULATORY AGENT. Attenuated strains of pathogens will lackall or parts of virulence operons, may lack immune-stimulatory surfacemoieties (e.g. lipopolysaccharide for Gram-negative bacteria), and maycontain one or more nutrient auxotrophies. In specific embodiments, theengineered microbes are attenuated intracellular pathogens, such asavirulent strains of Listeria monocytogenes.

In some embodiments, the composition of the invention comprises one ormore types of microbe capable of producing butyrate in a mammaliansubject. Butyrate-producing microbes may be identified experimentally,such as by NMR or gas chromatography analyses of microbial products orcolorimetric assays (Rose I A. 1955. Methods Enzymol. Acetate kinase ofbacteria. 1: 591-5). Butyrate-producing microbes may also be identifiedcomputationally, such as by the identification of one or more enzymesinvolved in butyrate synthesis. Non-limiting examples of enzymes foundin butyrate-producing microbes include butyrate kinase,phosphotransbutyrylase, and butyryl CoA:acetate CoA transferase (LouisP., et al. 2004. Restricted Distribution of the Butyrate Kinase Pathwayamong Butyrate-Producing Bacteria from the Human Colon. J Bact. 186(7):2099-2106). Butyrate-producing strains include, but are not limited to,Faecalibacterium prausnitzii, Eubacterium spp., Butyrivibriofibrisolvens, Roseburia intestinalis, Clostridium spp., Anaerostipescaccae, and Ruminococcus spp. In some embodiments, the compositioncomprises two or more types of microbe, wherein at least two types ofmicrobe are capable of producing butyrate in a mammalian subject. Inother embodiments, the composition comprises two or more types ofmicrobe, wherein two or more types of microbe cooperate (i.e.,cross-feed) to produce an immunomodulatory SCFA (e.g., butyrate) in amammalian subject. In a preferred embodiment, the composition comprisesat least one type of microbe (e.g., Bifidobacterium spp.) capable ofmetabolizing a prebiotic, including but not limited to, inulin,inulin-type fructans, or oligofructose, such that the resultingmetabolic product may be converted by a second type of microbe (e.g., abutyrate-producing microbe such as Roseburia spp.) to animmunomodulatory SCFA such as butyrate (Falony G., et al. 2006.Cross-Feeding between Bifidobacterium longum BB536 andAcetate-Converting, Butyrate-Producing Colon Bacteria during Grown onOligofructose. Appl. Environ. Microbiol. 72(12): 7835-7841.) In otheraspects, the composition comprises at least one acetate-producingmicrobe (e.g., Bacteroides thetaiotaomicron) and at least oneacetate-consuming, butyrate-producing microbe (e.g., Faecalibacteriumprausnitzii).

In some embodiments, the composition comprises one or more types ofmicrobe capable of producing propionate in a mammalian subject,optionally further comprising a prebiotic or substrate appropriate forproprionate biosynthesis. Examples of prebiotics or substrates used forthe production of propionate include, but are not limited to,L-rhamnose, D-tagalose, resistant starch, inulin, polydextrose,arabinoxylans, arabinoxylan oligosaccharides, mannooligosaccharides, andlaminarans (Hosseini E., et al. 2011. Propionate as a health-promotingmicrobial metabolite in the human gut. Nutrition Reviews. 69(5):245-258). Propionate-producing microbes may be identifiedexperimentally, such as by NMR or gas chromatography analyses ofmicrobial products or colorimetric assays (Rose I A. 1955. MethodsEnzymol. Acetate kinase of bacteria. 1: 591-5). Propionate-producingmicrobes may also be identified computationally, such as by theidentification of one or more enzymes involved in propionate synthesis.Non-limiting examples of enzymes found in propionate-producing microbesinclude enzymes of the succinate pathway, including but not limited tophophoenylpyrvate carboxykinase, pyruvate kinase, pyruvate carboxylase,malate dehydrogenase, fumarate hydratase, succinate dehydrogenase,succinyl CoA synthetase, methylmalonyl Coa decarboxylase, and propionateCoA transferase, as well as enzymes of the acrylate pathway, includingbut not limited to L-lactate dehydrogenase, propionate CoA transferase,lactoyl CoA dehydratase, acyl CoA dehydrogenase, phosphateacetyltransferase, and propionate kinase. Non-limiting examples ofmicrobes that utilize the succinate pathway are Bacteroides fragilis andother species (including B. vulgatus), Propionibacterium spp. (includingfreudenrichii and acidipropionici), Veillonella spp. (includinggazogenes), Micrococcus lactilyticus, Selenomonas ruminantium,Escherichia coli, and Prevotella ruminocola. Non-limiting examples ofmicrobes that utilize the acrylate pathway are Clostridiumneopropionicum X4, and Megasphaera elsdenii.

In preferred embodiments, the combination of a microbe or microbialcomposition and a prebiotic is selected based on the fermentation ormetabolic preferences of one or more microbes capable of producingimmunomodulatory SCFAs (e.g., preference for complex versus simple sugaror preference for a fermentation product versus a prebiotic). Forexample, M. eldsenii prefers lactate fermentation to glucosefermentation, and maximization of propionate production by M. eldseniiin a mammalian subject may therefore be achieved by administering alongwith M. eldsenii a favored substrate (e.g., lactate) or one or moremicrobes capable of fermenting glucose into lactate (e.g., Streptococcusbovis) (Hosseini E., et al. 2011. Propionate as a health-promotingmicrobial metabolite in the human gut. Nutrition Reviews. 69(5):245-258). Thus, in some embodiments, the composition comprises at leastone type of SCFA-producing microbe and a sugar fermentation product(e.g., lactate). In other embodiments, the composition comprises atleast one type of SCFA-producing microbe and at least one type ofsugar-fermenting microbe, wherein the fermentation product of thesecond, sugar-fermenting microbe is the preferred substrate of theSCFA-producing microbe.

Immunomodulation can also be achieved by the microbial production ofglutathione or gamma-glutamylcysteine. Thus, in certain embodiments, thepharmaceutical composition, dosage form, or kit comprises at least onetype of microbe capable of producing glutathione and/orgamma-glutamylcysteine in a mammalian subject. In some aspects, thecomposition comprises one or more microbes selected for the presence ofglutamate cysteine ligase (e.g., Lactobacillus fermentum) and/orL-proline biosynthesis enzymes (e.g., E. coli) (Peran et al., 2006.Lactobacillus fermenum, a probiotic capable to release glutathione,prevents colonic inflammation in the TNBS model of rat colitis. Int JColorectal Dis. 21(8): 737-746; Veeravalli et al., 2011. Laboratoryevolution of glutathione biosynthesis reveals naturally compensatorypathways. Nat Chem Bio. 7(2): 101-105). In a preferred embodiment, atleast one microbe in the composition is L. fermentum.

para-cresol (p-cresol) is a microbial product, via the fermentation oftyrosine or phenylalanine. Sulfated in the liver or colon to p-cresylsulfate, this molecule reduces Th1-mediated responses (Shiba T. et al.2014. Effects of intestinal bacteria-derived p-cresyl sulfate onTh1-type immune response in vivo and in vitro. Tox and Applied Pharm.274(2): 191-199). In some embodiments, the composition comprises atleast one type of microbe capable of fermenting tyrosine and/orphenylalanine to p-cresol in a mammalian subject. Non-limiting examplesof such microbes include Bacteroides fragilis, Clostridium difficile,and Lactobacillus sp. Strain #11198-11201 (Yokoyama M T and Carlson J R.1981. Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.Applied and Environmental Microbiology. 41(1): 71-76.), and othermicrobes with p-hydroxylphenyl acetate decarboxylase activity.

IV. Methods of Making/Isolating Probiotic Compositions

In one embodiment, provided herein are therapeutic compositionscontaining a purified population of bacterial entities and/or fungalentities. The purified population can contain a single species, ormultiple species. As used herein, the terms “purify”, “purified” and“purifying” refer to the state of a population (e.g., a plurality ofknown or unknown amount and/or concentration) of desired bacterialentities and/or fungal entities, that have undergone one or moreprocesses of purification, e.g., a selection or an enrichment of thedesired bacterial, or alternatively a removal or reduction of residualhabitat products as described herein. In some embodiments, a purifiedpopulation has no detectable undesired activity or, alternatively, thelevel or amount of the undesired activity is at or below an acceptablelevel or amount. In other embodiments, a purified population has anamount and/or concentration of desired bacterial entities and/or fungalentities at or above an acceptable amount and/or concentration. In otherembodiments, the ratio of desired-to-undesired activity (e.g., sporescompared to vegetative bacteria), has changed by 2-, 5-, 10-, 30-, 100-,300-, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, or greater than 1×10⁸. In otherembodiments, the purified population of bacterial entities and/or fungalentities is enriched as compared to the starting material (e.g., a fecalmaterial) from which the population is obtained. This enrichment may beby 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,99.9%, 99.99%, 99.999%, 99.9999%, 99.9999%, or greater than 99.999999%as compared to the starting material.

In certain embodiments, the purified populations of bacterial entitiesand/or fungal entities have reduced or undetectable levels of one ormore pathogenic activities, such as toxicity, an ability to causeinfection of the mammalian recipient subject, an undesiredimmunomodulatory activity, an autoimmune response, a metabolic response,or an inflammatory response or a neurological response. Such a reductionin a pathogenic activity may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, orgreater than 99.9999% as compared to the starting material. In otherembodiments, the purified populations of bacterial entities and/orfungal entities have reduced sensory components as compared to fecalmaterial, such as reduced odor, taste, appearance, and umami.

In another embodiment, the invention provides purified populations ofbacterial entities and/or fungal entities that are substantially free ofresidual habitat products. In certain embodiments, this means that thebacterial composition no longer contains a substantial amount of thebiological matter associated with the microbial community while livingon or in the human or animal subject, and the purified population ofspores may be 100% free, 99% free, 98% free, 97% free, 96% free, 95%free, 94% free, 93% free, 92% free, 91% free, 90% free, 85% free, 80%free, 75% free, 70% free, 60% free, or 50% free of any contamination ofthe biological matter associated with the microbial community.Substantially free of residual habitat products may also mean that thebacterial composition contains no detectable cells from a human oranimal, and that only microbial cells are detectable, in particular,only desired microbial cells are detectable. In another embodiment, itmeans that fewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%,1×10⁻⁷%, 1×10⁻⁸% of the cells in the bacterial composition are human oranimal, as compared to microbial cells. In another embodiment, theresidual habitat product present in the purified population is reducedat least a certain level from the fecal material obtained from themammalian donor subject, e.g., reduced by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%,99.999%, 99.9999%, or greater than 99.9999%.

In one embodiment, substantially free of residual habitat products orsubstantially free of a detectable level of a pathogenic material meansthat the bacterial composition contains no detectable viral (includingbacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmalcontaminants, or a eukaryotic parasite such as a helminth.Alternatively, the purified spore populations are substantially free ofan acellular material, e.g., DNA, viral coat material, or non-viablebacterial material. Alternatively, the purified spore population mayprocessed by a method that kills, inactivates, or removes one or morespecific undesirable viruses, such as an enteric virus, includingnorovirus, poliovirus or hepatitis A virus.

As described herein, purified spore populations can be demonstrated by,for example, genetic analysis (e.g., PCR, DNA sequencing), serology andantigen analysis, microscopic analysis, microbial analysis includinggermination and culturing, or methods using instrumentation such as flowcytometry with reagents that distinguish desired bacterial entitiesand/or fungal entities from non-desired, contaminating materials.

In one embodiment, the spore preparation comprises spore-forming specieswherein residual non-spore forming species have been inactivated bychemical or physical treatments including ethanol, detergent, heat,sonication, and the like; or wherein the non-spore forming species havebeen removed from the spore preparation by various separations stepsincluding density gradients, centrifugation, filtration and/orchromatography; or wherein inactivation and separation methods arecombined to make the spore preparation. In yet another embodiment, thespore preparation comprises spore-forming species that are enriched overviable non-spore formers or vegetative forms of spore formers. In thisembodiment, spores are enriched by 2-fold, 5-fold, 10-fold, 50-fold,100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared toall vegetative forms of bacteria. In yet another embodiment, the sporesin the spore preparation undergo partial germination during processingand formulation such that the final composition comprises spores andvegetative bacteria derived from spore forming species.

In another embodiment, provided herein are methods for production of acomposition, e.g., a probiotic composition, comprising a bacterialpopulation, e.g., an anti-inflammatory bacterial population, or a fungalpopulation, with or without one or more prebiotic, suitable fortherapeutic administration to a mammalian subject in need thereof. Inone embodiment, the composition can be produced by generally followingthe steps of: (a) providing a fecal material obtained from a mammaliandonor subject; and (b) subjecting the fecal material to at least onepurification treatment or step under conditions such that a populationof bacterial entities and/or fungal entities is produced from the fecalmaterial.

Individual bacterial strains can also be isolated from stool samplesusing culture methods. For example, 5 mls of phosphate-buffered saline(PBS) is added to 1 mg of frozen stool sample and homogenized byvortexing in an anaerobic chamber for isolation of anaerobic bacteria.The suspension is then serially diluted ten-fold (e.g. 10⁻¹ to 10⁻⁹dilutions) and 100 □l aliquots of each dilution are spread evenly overthe surface of agar plates containing different formulations e.g.anaerobic blood agar plates, Bacteroides bile esculin plates, lakedkanamycin vancomycin plates, egg yolk agar plates and de Man Rogosa andSharpe agar plates. Inverted plates are incubated in an anaerobicchamber for 48 hr+/−4 hours. Colonies with different morphologies arepicked and replated on anaerobic blood agar plates for further testing,PCR analysis and 16 S sequencing. Selected bacterial strains can begrown for therapeutic use singly or in combination.

In one embodiment, a probiotic composition of the invention is not afecal transplant. In some embodiments all or essentially all of thebacterial entitites present in a purified population are originallyobtained from a fecal material and subsequently, e.g., for production ofpharmaceutical compositions, are grown in culture as described herein orotherwise known in the art. In one embodiment, the bacterial cells arecultured from a bacterial stock and purified as described herein. In oneembodiment, each of the populations of bacterial cells are independentlycultured and purified, e.g., each population is cultured separately andsubsequently mixed together. In one embodiment, one or more of thepopulations of bacterial cells in the composition are co-cultured.

Donor Materials and Screening

Typically, bacteria and fungi are derived from biological samples, whichmay include one or more micriobiotal populations. Exemplary biologicalsamples include fecal materials such as feces or materials isolated fromthe various segments of the small and large intestine. Fecal materialsare obtained from a mammalian donor subject, or can be obtained frommore than one donor subject, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or fromgreater than 1000 donors, where such materials are then pooled prior topurification of the desired bacterial entities and/or fungal entities.In another embodiment, fecal materials can be obtained from a singledonor subject over multiple times and pooled from multiple samples,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 32, 35, 40, 45, 48,50, 100 samples from a single donor.

In alternative embodiments, the desired bacterial entities and/or fungalentities are purified from a single fecal material sample obtained froma single donor, and after such purification are combined with purifiedspore populations from other purifications, either from the same donorat a different time, or from one or more different donors, or both.

In some embodiments, all or essentially all of the bacterial entitiesand/or fungal entities present in a purified population are obtainedfrom a fecal material treated as described herein or otherwise known inthe art. In some embodiments all or essentially all of the bacterialentitites and/or fungal entities present in a purified population areobtained from a fecal material and subsequently are grown in culture asdescribed herein or otherwise known in the art. In alternativeembodiments, one or more than one bacterial entities and/or fungalentities or types of bacterial entities and/or fungal entities aregenerated in culture and combined to form a purified spore population.In other alternative embodiments, one or more of these culture-generatedspore populations are combined with a fecal material-derived sporepopulation to generate a hybrid spore population.

Preferably the biological sample includes a fecal material, such asobtained from a healthy mammalian donor subject or a plurality ofmammalian donor subjects. In some embodiments, the biological materialis not a fecal sample. Other appropriate biological samples include, butare not limited to, vaginal or cervical swabs, skin swabs, andbronchoalveolar lavage fluid (BALF).

In some embodiments, mammalian donor subjects are generally of goodhealth and have microbiota consistent with such good health. In oneembodiment, the donor subjects have not been administered antibioticcompounds within a certain period prior to the collection of the fecalmaterial. In certain embodiments, the donor subjects are not obese oroverweight, and may have body mass index (BMI) scores of below 25, suchas between 18.5 and 24.9. In other embodiments, the donor subjects arenot mentally ill or have no history or familial history of mentalillness, such as anxiety disorder, depression, bipolar disorder, autismspectrum disorders, schizophrenia, panic disorders, attention deficit(hyperactivity) disorders, eating disorders or mood disorders. In otherembodiments, the donor subjects do not have Irritable Bowel Disease(e.g., crohn's disease, ulcerative colitis), irritable bowel syndrome,celiac disease, colorectal cancer or a family history of these diseases.In other embodiments, donors have been screened for blood bornepathogens and fecal transmissible pathogens using standard techniquesknown to one in the art (e.g., nucleic acid testing, serologicaltesting, antigen testing, culturing techniques, enzymatic assays, assaysof cell free fecal filtrates looking for toxins on susceptible cellculture substrates).

In some embodiments, donors are also selected for the presence ofcertain genera and/or species that provide increased efficacy oftherapeutic compositions containing these genera or species. In otherembodiments, donors are preferred that produce relatively higherconcentrations of spores in fecal material than other donors. In furtherembodiments, donors are preferred that provide fecal material from whichspores having increased efficacy are purified; this increased efficacyis measured using in vitro or in animal studies as described below. Insome embodiments, the donor may be subjected to one or more pre-donationtreatments in order to reduce undesired material in the fecal material,and/or increase desired spore populations.

In one embodiment, it is advantageous to screen the health of the donorsubject prior to and optionally, one or more times after, the collectionof the fecal material. Such screening identifies donors carryingpathogenic materials such as viruses (HIV, hepatitis, polio) andpathogenic bacteria. Post-collection, donors are screened about oneweek, two weeks, three weeks, one month, two months, three months, sixmonths, one year or more than one year, and the frequency of suchscreening may be daily, weekly, bi-weekly, monthly, bi-monthly,semi-yearly or yearly. Donors that are screened and do not testpositive, either before or after donation or both, are considered“validated” donors.

Methods for Purifying Spores

In one embodiment, treatment of fecal sample includes heating thematerial, e.g., above 25 degrees Celsius for at least 30 seconds, and/orcontacting the material with a solvent, and/or and or contacting achemical or providing a physical manipulation of the material. Cultureof fecal material includes replicating the purified population in aliquid suspension and/or a solid medium. Optionally, one removes atleast a portion of an acellular component of the fecal material, therebyseparating immunomodulatory bacteria from acellular material. Thetreatment step may also include depleting or inactivating a pathogenicmaterial.

Solvent Treatments.

The bacteria and/or fungi may contain a purified population obtainedfrom a miscible solvent treatment of the fecal material or a fraction orderivative thereof. In one embodiment, to purify the bacterial entitiesand/or fungal entities, the fecal material can be subjected to one ormore solvent treatments. A solvent treatment is a miscible solventtreatment (either partially miscible or fully miscible) or an immisciblesolvent treatment. Miscibility is the ability of two liquids to mix witheach to form a homogeneous solution. Water and ethanol, for example, arefully miscible such that a mixture containing water and ethanol in anyratio will show only one phase. Miscibility is provided as a wt/wt %, orweight of one solvent in 100 g of final solution. If two solvents arefully miscible in all proportions, their miscibility is 100%. Providedas fully miscible solutions with water are alcohols, e.g., methanol,ethanol, isopropanol, butanol, propanediol, butanediol, etc. Thealcohols can be provided already combined with water; e.g., a solutioncontaining 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 89%, 85%, 90%, 95% or greater than 95% water. Other solvents areonly partially miscible, meaning that only some portion will dissolve inwater. Diethyl ether, for example, is partially miscible with water. Upto 7 grams of diethyl ether will dissolve in 93 grams of water to give a7% (wt/wt %) solution. If more diethyl ether is added, a two-phasesolution will result with a distinct diethyl ether layer above thewater. Other partially miscible materials include ethers, propanoate,butanoate, chloroform, dimethoxyethane, or tetrahydrofuran. In contrast,an oil such as an alkane and water are immiscible and form two phases.Further, immiscible treatments are optionally combined with a detergent,either an ionic detergent or a non-ionic detergent. Exemplary detergentsinclude Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or apolyol.

In one embodiment, the solvent treatment steps reduces the viability ofnon-spore forming bacterial species by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, andit may optionally reduce the viability of contaminating protists,parasites and/or viruses.

Chromatography Treatments.

To purify spore populations, the fecal materials may be subjected to oneor more chromatographic treatments, either sequentially or in parallel.In a chromatographic treatment, a solution containing the fecal materialis contacted with a solid medium containing a hydrophobic interactionchromatographic (HIC) medium or an affinity chromatographic medium. Inan alternative embodiment, a solid medium capable of absorbing aresidual habitat product present in the fecal material is contacted witha solid medium that adsorbs a residual habitat product. In certainembodiments, the HIC medium contains sepharose or a derivatizedsepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, orbutyl-s sepharose. In other embodiments, the affinity chromatographicmedium contains material derivatized with mucin type I, II, III, IV, V,or VI, or oligosaccharides derived from or similar to those of mucinstype I, II, III, IV, V, or VI. Alternatively, the affinitychromatographic medium contains material derivatized with antibodiesthat recognize immunomodulatory bacteria.

Mechanical Treatments.

In one embodiment, the fecal material can be physically disrupted,particularly by one or more mechanical treatment such as blending,mixing, shaking, vortexing, impact pulverization, and sonication. Asprovided herein, the mechanical disrupting treatment substantiallydisrupts a non-spore material present in the fecal material and does notsubstantially disrupt a spore present in the fecal material, or it maydisrupt the spore material less than the non-spore material, e.g.,2-fold less, 5-, 10-, 30-, 100-, 300-, 1000- or greater than 1000-foldless. Furthermore, mechanical treatment homogenizes the material forsubsequent sampling, testing, and processing. Mechanical treatmentsoptionally include filtration treatments, where the desired sporepopulations are retained on a filter while the undesirable (non-spore)fecal components to pass through, and the spore fraction is thenrecovered from the filter medium. Alternatively, undesirableparticulates and eukaryotic cells may be retained on a filter whilebacterial cells including spores pass through. In some embodiments thespore fraction retained on the filter medium is subjected to adiafiltration step, wherein the retained spores are contacted with awash liquid, typically a sterile saline-containing solution or otherdiluent such as a water compatible polymer including a low-molecularpolyethylene glycol (PEG) solution, in order to further reduce or removethe undesirable fecal components.

Thermal Treatments.

In another embodiment, thermal disruption of the fecal material may beutilized. Generally, in one embodiment, the fecal material is mixed in asaline-containing solution such as phosphate-buffered saline (PBS) andsubjected to a heated environment, such as a warm room, incubator,water-bath, or the like, such that efficient heat transfer occursbetween the heated environment and the fecal material. Preferably thefecal material solution is mixed during the incubation to enhancethermal conductivity and disrupt particulate aggregates. Thermaltreatments can be modulated by the temperature of the environment and/orthe duration of the thermal treatment. For example, the fecal materialor a liquid comprising the fecal material is subjected to a heatedenvironment, e.g., a hot water bath of at least about 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or greater than 100degrees Celsius, for at least about 1, 5, 10, 15, 20, 30, 45 seconds, or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 hours. In certain embodimentsthe thermal treatment occurs at two different temperatures, such as 30seconds in a 100 degree Celsius environment followed by 10 minutes in a50 degree Celsius environment. In preferred embodiments the temperatureand duration of the thermal treatment are sufficient to kill or removepathogenic materials while not substantially damaging or reducing thegermination-competency of the spores. In other preferred embodiments,the temperature and duration of the thermal treatment is short enough toreduce the germination of the spore population.

Irradiation Treatments.

In another embodiment, methods of treating the fecal material orseparated contents of the fecal material with ionizing radiation,typically gamma irradiation, ultraviolet irradiation or electron beamirradiation provided at an energy level sufficient to kill pathogenicmaterials while not substantially damaging the desired spore populationsmay be used. For example, ultraviolet radiation at 254 nm provided at anenergy level below about 22,000 microwatt seconds per cm² will notgenerally destroy desired spores.

Centrifugation and Density Separation Treatments.

In one embodiment, desired spore populations may be separated from theother components of the fecal material by centrifugation. For example, asolution containing the fecal material can be subjected to one or morecentrifugation treatments, e.g., at about 200×g, 1000×g, 2000×g, 3000×g,4000×g, 5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g.Differential centrifugation separates desired spores from undesirednon-spore material; at low forces the spores are retained in solution,while at higher forces the spores are pelleted while smaller impurities(e.g., virus particles, phage, microscopic fibers, biologicalmacromolecules such as free protein, nucleic acids and lipids) areretained in solution. For example, a first low force centrifugationpellets fibrous materials; a second, higher force centrifugation pelletsundesired eukaryotic cells, and a third, still higher forcecentrifugation pellets the desired spores while smaller contaminantsremain in suspension. In some embodiments density or mobility gradientsor cushions (e.g., step cushions), such as CsCl, Percoll, Ficoll,Nycodenz, Histodenz or sucrose gradients, are used to separate desiredspore populations from other materials in the fecal material.

Also provided herein are methods of producing spore populations thatcombine two or more of the treatments described herein in order tosynergistically purify the desired spores while killing or removingundesired materials and/or activities from the spore population. It isgenerally desirable to retain the spore populations undernon-germinating and non-growth promoting conditions and media, in orderto minimize the growth of pathogenic bacteria present in the sporepopulations and to minimize the germination of spores into vegetativebacterial cells.

The bacteria and/or fungi may contain a spore population, e.g., sporesand/or spore-formers, or a population containing vegetative cells.

Methods for Preparing a Bacterial Composition for Administration to aSubject.

In one embodiment, methods for producing bacterial compositions caninclude three main processing steps, combined with one or more mixingsteps. For example, the steps can include organism banking, organismproduction, and preservation.

For banking, the strains included in the bacterial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments that use a culturing step, the agar or broth can containnutrients that provide essential elements and specific factors thatenable growth. An example includes a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. A variety of microbiological media and variations arewell known in the art (e.g. R. M. Atlas, Handbook of MicrobiologicalMedia (2010) CRC Press). Medium can be added to the culture at thestart, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of thebacterial composition, or as an entire collection comprising thebacterial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture may be incubated under favorable conditions for atime sufficient to build biomass. For bacterial compositions for humanuse, this may be at 37° C., with pH, and other parameters having valuessimilar to the normal human niche. The environment can be activelycontrolled, passively controlled (e.g., via buffers), or allowed todrift. For example, for anaerobic bacterial compositions (e.g., gutmicrobiota), an anoxic/reducing environment can be employed. This can beaccomplished by addition of reducing agents such as cysteine to thebroth, and/or stripping it of oxygen. As an example, a culture of abacterial composition can be grown at 37° C., pH 7, in the medium above,pre-reduced with 1 g/L cysteine-HCl.

In one embodiment, when the culture has generated sufficient biomass, itcan be preserved for banking. The organisms can be placed into achemical milieu that protects from freezing (adding ‘cryoprotectants’),drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’),dispensing into multiple (optionally identical) containers to create auniform bank, and then treating the culture for preservation. In oneembodiment, containers can be generally impermeable and have closuresthat assure isolation from the environment. Cryopreservation treatmentcan be accomplished by freezing a liquid at ultra-low temperatures(e.g., at or below −80° C.). Dried preservation removes water from theculture by evaporation (in the case of spray drying or ‘cool drying’) orby sublimation (e.g., for freeze drying, spray freeze drying). Removalof water improves long-term bacterial composition storage stability attemperatures elevated above cryogenic. If the bacterial compositioncomprises spore forming species and results in the production of spores,the final composition can be purified by additional means, such asdensity gradient centrifugation preserved using the techniques describedabove. Bacterial composition banking can be done by culturing andpreserving the strains individually, or by mixing the strains togetherto create a combined bank. As an example of cryopreservation, abacterial composition culture can be harvested by centrifugation topellet the cells from the culture medium, the supernate decanted andreplaced with fresh culture broth containing 15% glycerol. The culturecan then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C.for long-term viability retention. This procedure achieves acceptableviability upon recovery from frozen storage.

Organism production can be conducted using similar culture steps tobanking, including medium composition and culture conditions. In oneembodiment, it can be conducted at larger scales of operation,especially for clinical development or commercial production. At largerscales, there can be several subcultivations of the bacterialcomposition prior to the final cultivation. At the end of cultivation,the culture can be harvested to enable further formulation into a dosageform for administration. This can involve concentration, removal ofundesirable medium components, and/or introduction into a chemicalmilieu that preserves the bacterial composition and renders itacceptable for administration via the chosen route. For example, abacterial composition can be cultivated to a concentration of 10¹⁰CFU/mL, then concentrated 20-fold by tangential flow microfiltration;the spent medium can be exchanged by diafiltering with a preservativemedium consisting of s2% gelatin, 100 mM trehalose, and 10 mM sodiumphosphate buffer. The suspension can then be freeze-dried to a powderand titrated.

In one embodiment, after drying, the powder can be blended to anappropriate potency, and mixed with other cultures and/or a filler suchas microcrystalline cellulose for consistency and ease of handling, andthe bacterial composition formulated as provided herein.

Methods of Characterization of Compositions

In certain embodiments, methods are provided for testing certaincharacteristics of compositions comprising microbes or microbes andprebiotics. For example, the sensitivity of bacterial compositions tocertain environmental variables is determined, e.g., in order to selectfor particular desirable characteristics in a given composition,formulation and/or use. For example, the bacterial constituents of thecomposition can be tested for pH resistance, bile acid resistance,and/or antibiotic sensitivity, either individually on aconstituent-by-constituent basis or collectively as a bacterialcomposition comprised of multiple bacterial constituents (collectivelyreferred to in this section as bacterial composition).

pH Sensitivity Testing.

If a microbial composition, with or without prebiotic, will beadministered other than to the colon or rectum (i.e., for example, anoral route), optionally testing for pH resistance enhances the selectionof microbes or therapeutic compositions that will survive at the highestyield possible through the varying pH environments of the distinctregions of the GI tract or vagina. Understanding how the bacterialcompositions react to the pH of the GI tract or vagina also assists informulation, so that the number of microbes in a dosage form can beincreased if beneficial and/or so that the composition may beadministered in an enteric-coated capsule or tablet or with a bufferingor protective composition.

As the pH of the stomach can drop to a pH of 1 to 2 after a high-proteinmeal for a short time before physiological mechanisms adjust it to a pHof 3 to 4 and often resides at a resting pH of 4 to 5, and as the pH ofthe small intestine can range from a pH of 6 to 7.4, bacterialcompositions can be prepared that survive these varying pH ranges(specifically wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or as much as 100% of the bacteria can survivegut transit times through various pH ranges). This can be tested byexposing the bacterial composition to varying pH ranges for the expectedgut transit times through those pH ranges. Therefore, as a nonlimitingexample only, 18-hour cultures of compositions comprising one or morebacterial species or strains can be grown in standard media, such as gutmicrobiota medium (“GMM”, see Goodman et al., Extensive personal humangut microbiota culture collections characterized and manipulated ingnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or anotheranimal-products-free medium, with the addition of pH adjusting agentsfor a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. Analternative method for testing stability to acid is described in U.S.Pat. No. 4,839,281. Survival of bacteria may be determined by culturingthe bacteria and counting colonies on appropriate selective ornon-selective media.

Bile Acid Sensitivity Testing.

Additionally, in some embodiments, testing for bile-acid resistanceenhances the selection of microbes or therapeutic compositions that willsurvive exposures to bile acid during transit through the GI tract orvagina. Bile acids are secreted into the small intestine and can, likepH, affect the survival of bacterial compositions. This can be tested byexposing the compositions to bile acids for the expected gut exposuretime to bile acids. For example, bile acid solutions can be prepared atdesired concentrations using 0.05 mM Tris at pH 9 as the solvent. Afterthe bile acid is dissolved, the pH of the solution may be adjusted to7.2 with 10% HCl. Bacterial components of the therapeutic compositionscan be cultured in 2.2 ml of a bile acid composition mimicking theconcentration and type of bile acids in the patient, 1.0 ml of 10%sterile-filtered feces media and 0.1 ml of an 18-hour culture of thegiven strain of bacteria. Incubations may be conducted for from 2.5 to 3hours or longer. An alternative method for testing stability to bileacid is described in U.S. Pat. No. 4,839,281. Survival of bacteria maybe determined by culturing the bacteria and counting colonies onappropriate selective or non-selective media.

Antibiotic Sensitivity Testing.

As a further optional sensitivity test, the bacterial components of themicrobial compositions, with or without prebiotics, can be tested forsensitivity to antibiotics. In one embodiment, the bacterial componentscan be chosen so that they are sensitive to antibiotics such that ifnecessary they can be eliminated or substantially reduced from thepatient's gastrointestinal tract or vagina by at least one antibiotictargeting the bacterial composition.

Adherence to Gastrointestinal Cells.

The compositions may optionally be tested for the ability to adhere togastrointestinal cells. A method for testing adherence togastrointestinal cells is described in U.S. Pat. No. 4,839,281.

Identification of Immunomodulatory Bacteria.

In some embodiments, immunomodulatory bacteria are identified by thepresence of nucleic acid sequences that modulate sporulation. Inparticular, signature sporulation genes are highly conserved acrossmembers of distantly related genera including Clostridium and Bacillus.Traditional approaches of forward genetics have identified many, if notall, genes that are essential for sporulation (spo). The developmentalprogram of sporulation is governed in part by the successive action offour compartment-specific sigma factors (appearing in the order σF, σE,σG and σK), whose activities are confined to the forespore (σF and σG)or the mother cell (σE and σK). In other embodiments, immunomodulatorybacteria are identified by the biochemical activity of DPA producingenzymes or by analyzing DPA content of cultures. As part of thebacterial sporulation, large amounts of DPA are produced, and comprise5-15% of the mass of a spore. Because not all viable spores germinateand grow under known media conditions, it is difficult to assess a totalspore count in a population of bacteria. As such, a measurement of DPAcontent highly correlates with spore content and is an appropriatemeasure for characterizing total spore content in a bacterialpopulation.

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria induce secretion ofpro-inflammatory or anti-inflammatory cytokines by host cells. Forexample, human or mammalian cells capable of cytokine secretion, such asimmune cells (e.g., PBMCs, macrophages, T cells, etc.) can be exposed tocandidate immunomodulatory bacteria, or supernatants obtained fromcultures of candidate immunomodulatory bacteria, and changes in cytokineexpression or secretion can be measured using standard techniques, suchas ELISA, immunoblot, Luminex, antibody array, quantitative PCR,microarray, etc. Bacteria can be selected for inclusion in a probioticcomposition based on the ability to induce a desired cytokine profile inhuman or mammalian cells. For example, anti-inflammatory bacteria can beselected for inclusion in a probiotic composition based on the abilityto induce secretion of one or more anti-inflammatory cytokines, and/orthe ability to reduce secretion of one or more pro-inflammatorycytokines. Anti-inflammatory cytokines include, for example, IL-10,IL-13, IL-9, IL-4, IL-5, and combinations thereof. Other inflammatorycytokines include, for example, TGFβ. Pro-inflammatory cytokinesinclude, for example, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α,MIP1β, TNFα, and combinations thereof. In some embodiments,anti-inflammatory bacteria may be selected for inclusion in a probioticcomposition based on the ability to modulate secretion of one or moreanti-inflammatory cytokines and/or the ability to reduce secretion ofone or more pro-inflammatory cytokines by a host cell induced by abacteria of a different type (e.g., a bacteria from a different speciesor from a different strain of the same species).

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria impact thedifferentiation and/or expansion of particular subpopulations of immunecells. For example, candidate bacteria can be screened for the abilityto promote differentiation and/or expansion of Treg cells, Th17 cells,Th1 cells and/or Th2 cells from precursor cells, e.g. naive T cells. Byway of example, naïve T cells can be cultured in the presence ofcandidate bacteria or supernatants obtained from cultures of candidatebacteria, and numbers of Treg cells, Th17 cells, Th1 cells and/or Th2cells can be determined using standard techniques, such as FACSanalysis. Markers indicative of Treg cells include, for example,CD25⁺CD127^(lo). Markers indicative of Th17 cells include, for example,CXCR3⁻CCR6⁺. Markers indicative of Th1 cells include, for example,CXCR3⁺CCR6⁻. Markers indicative of Th2 cells include, for example,CXCR3⁻CCR6⁻. Other markers indicative of particular T cellssubpopulations are known in the art, and may be used in the assaysdescribed herein, e.g., to identify populations of immune cells impactedby candidate immunomodulatory bacteria. Bacteria can be selected forinclusion in a probiotic composition based on the ability to promotedifferentiation and/or expansion of a desired immune cell subpopulation.

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria secrete short chainfatty acids (SCFA), such as, for example, butyrate, acetate, propionate,or valerate, or combinations thereof. For example, secretion of shortchain fatty acids into bacterial supernatants can be measured usingstandard techniques. In one embodiment, bacterial supernatants can bescreened to measure the level of one or more short chain fatty acidsusing NMR, mass spectrometry (e.g., GC-MS, tandem mass spectrometry,matrix-assisted laser desorption/ionization, etc.), ELISA, orimmunoblot. Expression of bacterial genes responsible for production ofshort chain fatty acids can also be determined by standard techniques,such as Northern blot, microarray, or quantitative PCR.

V. Mixtures of Bacteria and Microbial Networks

In one embodiment, provided herein are spore populations containing morethan one type of bacterium. As used herein, a “type” or more than one“types” of bacteria may be differentiated at the genus level, thespecies level, the sub-species level, the strain level or by any othertaxonomic method, as described herein and otherwise known in the art.

In one embodiment, the microbial, e.g., probiotic, population comprisesa single microbial preparation or a combination of microbialpreparations, wherein each microbial preparation can be purified from afecal material obtained from a single mammalian donor subject, or fromtwo or more donor subjects.

In some embodiments, all or essentially all of the bacterial entitiesand/or fungal entities present in an isolated population are obtainedfrom a fecal material treated as described herein or otherwise known inthe art. In alternative embodiments, one or more than one bacterialentities and/or fungal entities or types of bacterial entities and/orfungal entities are generated in culture and combined to form a purifiedspore population. In other alternative embodiments, one or more of theseculture-generated spore populations are combined with one or more fecalmaterial-derived spore population to generate a hybrid spore population.

In a preferred embodiment, a bacterial, e.g., probiotic, composition maycontain one or at least two types of preferred bacteria, includingstrains of the same species or of different species. For instance, abacterial composition may comprise 1, at least 2, at least 3, or atleast 4 types of bacteria. In another embodiment, a bacterialcomposition may comprise at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, orat least 20 or more than 20 types of bacteria, as defined by species oroperational taxonomic unit (OTU) encompassing such species. In apreferred embodiment, a bacterial composition comprises from 2 to nomore than 40, from 2 to no more than 30, from 2 to no more than 20, from2 to no more than 15, from 2 to no more than 10, from 2 to no more than5, types of bacteria. In another preferred embodiment, a bacterialcomposition comprises a single type of bacteria.

In one embodiment, bacterial compositions may comprise two types ofbacteria (termed “binary combinations” or “binary pairs”) or greaterthan two types of bacteria. Bacterial compositions that comprise threetypes of bacteria are termed “ternary combinations”.

Microbial compositions can comprise two types of microbes or a largenumber of microbe types. As used herein, a “type” or more than one“types” of microbes may be differentiated at the genus level, thespecies level, the sub-species level, the strain level or by any othertaxonomic method, as described herein and otherwise known in the art.For instance, a microbial composition can comprise at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or at least 40, at least 50 or greater than 50 typesof microbes, e.g. as defined by species or operational taxonomic unit(OTU), or otherwise as provided herein. In some embodiments, themicrobial composition includes at least 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, or greater numbers of types ofmicrobes.

Alternatively, the number of types of microbes present in a microbialcomposition is at or below a known value. For example, the microbialcomposition comprises 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100,50 or fewer types of microbes, such as 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer, or 9or fewer types of microbes, 8 or fewer types of microbes, 7 or fewertypes of microbes, 6 or fewer types of microbes, 5 or fewer types ofmicrobes, 4 or fewer types of microbes, or 3 or fewer types of microbes.In a preferred embodiment, a bacterial composition comprises from 2 tono more than 40, from 2 to no more than 30, from 2 to no more than 20,from 2 to no more than 15, from 2 to no more than 10, from 2 to no morethan 5, types of microbes. In another preferred embodiment, a bacterialcomposition comprises a single type of microbe.

In a preferred embodiment, the composition comprises about 20 or fewerisolated populations of bacterial cells. In another embodiment, thecomposition comprises about 15 or fewer isolated populations ofbacterial cells. In another embodiment, the composition comprises about10 or fewer isolated populations of bacterial cells. In anotherembodiment, the composition comprises about 5 or fewer isolatedpopulations of bacterial cells. In another embodiment, the compositioncomprises about 4 or fewer isolated populations of bacterial cells. Inanother embodiment, the composition comprises about 3 or fewer isolatedpopulations of bacterial cells. In another embodiment, the compositioncomprises about 2 isolated populations of bacterial cells. In anotherembodiment, the composition comprises between about 12 and 20 isolatedpopulations of bacterial cells. In another embodiment, the compositioncomprises a single isolated population of bacterial cells. In anotherembodiment, the composition comprises at least two isolated populationsof bacterial cells. In yet another embodiment, the composition comprisesabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 isolated populations of bacterial cells.

Aspects of the invention relate to microbial compositions that arereconstituted from purified strains. Provided are microbial compositionscomprising at least one, at least two or at least three microbes thatare not identical and that are capable of decreasing the risk and/orseverity of an autoimmune or inflammatory disease, symptom, condition,or disorder, or dysbiosis. In an embodiment, the microbial compositioncomprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 types of isolatedmicrobes. In one embodiment, the microbial composition comprises atleast about 4 types of isolated microbes or at least about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more types of isolated microbes. In some embodiments,the above invention relates to microbial compositions further comprisingone or more prebiotics.

Bacterial Compositions can be Described by Operational Taxonomic Units(OTUs).

Bacterial compositions may be prepared comprising one or at least twotypes of isolated bacteria, wherein a first type and a second type areindependently chosen from the species or OTUs listed in Table 1. Certainembodiments of bacterial compositions with at least two types ofisolated bacteria containing binary pairs are reflected herein.Additionally, a bacterial composition may be prepared comprising atleast two types of isolated bacteria, wherein a first OTU and a secondOTU are independently characterized by, i.e., at least 95%, 96%, 97%,98%, 99% or including 100% sequence identity to, sequences listed.

Bacterial compositions may be prepared comprising one or at least twotypes of isolated bacteria, chosen from the species in Table 1, Table1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, or Table 5.Generally, the first bacteria and the second bacteria are not the same.The sequences provided in the sequencing listing file for OTUs in Table1 are full 16S sequences. Therefore, in one embodiment, the first and/orsecond OTUs may be characterized by the full 16S sequences of OTUslisted in Table 1. In another embodiment, the first and/or second OTUsmay be characterized by one or more of the variable regions of the 16Ssequence (V1-V9). These regions in bacteria are defined by nucleotides69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173,1243-1294 and 1435-1465 respectively using numbering based on the E.coli system of nomenclature. (See, e.g., Brosius et al., Completenucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli,PNAS 75(10):4801-4805 (1978)). In some embodiments, at least one of theV1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterizean OTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU.

OTUs may be defined either by full 16S sequencing of the rRNA gene, bysequencing of a specific hypervariable region of this gene (i.e., V1,V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combinationof hypervariable regions from this gene (e.g. V1-3 or V3-5). Thebacterial 16S rDNA is approximately 1500 nucleotides in length and isused in reconstructing the evolutionary relationships and sequencesimilarity of one bacterial isolate to another using phylogeneticapproaches. 16S sequences are used for phylogenetic reconstruction asthey are in general highly conserved, but contain specific hypervariableregions that harbor sufficient nucleotide diversity to differentiategenera and species of most microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S sequence, genomicDNA is extracted from a bacterial sample, the 16S rDNA (full region orspecific hypervariable regions) amplified using polymerase chainreaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingmay be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

OTUs can be defined by a combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof, full-genome sequence, or partial genome sequencegenerated using amplified genetic products, or whole genome sequence(WGS). Using well defined methods DNA extracted from a bacterial samplewill have specific genomic regions amplified using PCR and sequenced todetermine the nucleotide sequence of the amplified products. In thewhole genome shotgun (WGS) method, extracted DNA will be directlysequenced without amplification. Sequence data can be generated usingany sequencing technology including, but not limited to Sanger,Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences,and/or Oxford Nanopore.

VI. Prebiotic Compositions

A prebiotic allows specific changes, both in the composition and/oractivity in the gastrointestinal microbiota, that confers benefits uponhost well-being and health. Prebiotics can include complexcarbohydrates, amino acids, peptides, or other nutritional componentsuseful for the survival of the bacterial composition. Prebioticsinclude, but are not limited to, amino acids, biotin,fructooligosaccharide, galactooligosaccharides, inulin, lactulose,mannan oligosaccharides, oligofructose-enriched inulin, oligofructose,oligodextrose, tagatose, trans-galactooligosaccharide, andxylooligosaccharides.

Suitable prebiotics are usually plant-derived complex carbohydrates,oligosaccharides or polysaccharides. Generally, prebiotics areindigestible or poorly digested by humans and serve as a food source forbacteria. Prebiotics which can be used in the pharmaceutical dosageforms, pharmaceutical compositions, and kits provided herein include,without limitation, galactooligosaccharides (GOS),trans-galactooligosaccharides, fructooligosaccharides or oligofructose(FOS), inulin, oligofructose-enriched inulin, lactulose, arabinoxylan,xylooligosaccharides (XOS), mannooligosaccharides, gum guar, gum Arabic,tagatose, amylose, amylopectin, xylan, pectin, and the like andcombinations of thereof. Prebiotics can be found in certain foods, e.g.chicory root, Jerusalem artichoke, Dandelion greens, garlic, leek,onion, asparagus, wheat bran, wheat flour, banana, milk, yogurt,sorghum, burdock, broccoli, Brussels sprouts, cabbage, cauliflower,collard greens, kale, radish and rutabaga, and miso. Alternatively,prebiotics can be purified or chemically or enzymatically synthesized.

Prebiotics of the Invention

In some embodiments, the composition comprises at least one prebiotic.In one embodiment, the prebiotic is a carbohydrate. In some embodiments,the composition of the present invention comprises a prebiotic mixture,which comprises at least one carbohydrate. A “carbohydrate” refers to asugar or polymer of sugars. The terms “saccharide,” “polysaccharide,”“carbohydrate,” and “oligosaccharide” may be used interchangeably. Mostcarbohydrates are aldehydes or ketones with many hydroxyl groups,usually one on each carbon atom of the molecule. Carbohydrates generallyhave the molecular formula (CH₂O)n. A carbohydrate can be amonosaccharide, a disaccharide, trisaccharide, oligosaccharide, orpolysaccharide. The most basic carbohydrate is a monosaccharide, such asglucose, sucrose, galactose, mannose, ribose, arabinose, xylose, andfructose. Disaccharides are two joined monosaccharides. Exemplarydisaccharides include sucrose, maltose, cellobiose, and lactose.Typically, an oligosaccharide includes between three and sixmonosaccharide units (e.g., raffinose, stachyose), and polysaccharidesinclude six or more monosaccharide units. Exemplary polysaccharidesinclude starch, glycogen, and cellulose. Carbohydrates can containmodified saccharide units, such as 2′-deoxyribose wherein a hydroxylgroup is removed, 2′-fluororibose wherein a hydroxyl group is replacewith a fluorine, or N-acetylglucosamine, a nitrogen-containing form ofglucose (e.g., 2′-fluororibose, deoxyribose, and hexose). Carbohydratescan exist in many different forms, for example, conformers, cyclicforms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.Carbohydrates may be purified from natural (e.g., plant or microbial)sources (i.e., they are enzymatically synthetized), or they may bechemically synthesized or modified. Such prebiotics can optionally beused in conjunction with one or more probiotics in the compositions andmethods of the invention. Exemplary prebiotics are provided in Table 7.

Suitable prebiotic carbohydrates can include one or more of acarbohydrate, carbohydrate monomer, carbohydrate oligomer, orcarbohydrate polymer. In certain embodiments, the pharmaceuticalcomposition, dosage form, or kit comprises at least one type of microbeand at least one type of non-digestible saccharide, which includesnon-digestible monosaccharides, non-digestible oligosaccharides, ornon-digestible polysaccharides. In one embodiment, the sugar units of anoligosaccharide or polysaccharide can be linked in a single straightchain or can be a chain with one or more side branches. The length ofthe oligosaccharide or polysaccharide can vary from source to source. Inone embodiment, small amounts of glucose can also be contained in thechain. In another embodiment, the prebiotic composition can be partiallyhydrolyzed or contain individual sugar moieties that are components ofthe primary oligosaccharide (see U.S. Pat. No. 8,486,668, PREBIOTICFORMULATIONS AND METHODS OF USE).

Prebiotic carbohydrates may include, but are not limited tomonosaccharaides (e.g., trioses, tetroses, pentoses, aldopentoses,ketopentoses, hexoses, cyclic hemiacetals, ketohexoses, heptoses) andmultimers thereof, as well as epimers, cyclic isomers, stereoisomers,and anomers thereof. Nonlimiting examples of monosaccharides include (ineither the L- or D-conformation) glyceraldehyde, threose, ribose,altrose, glucose, mannose, talose, galactose, gulose, idose, lyxose,arabanose, xylose, allose, erythrose, erythrulose, tagalose, sorbose,ribulose, psicose, xylulose, fructose, dihydroxyacetone, and cyclic(alpha or beta) forms thereof. Multimers (disaccharides, trisaccharides,oligosaccharides, polysaccharides) thereof include but are not limitedto sucrose, lactose, maltose, lactulose, trehalose, cellobiose,kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentioboise,turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiulose,rutinose, rutinulose, xylobiose, primeverose, amylose, amylopectin,starch (including resistant starch), chitin, cellulose, agar, agarose,xylan, glycogen, bacterial polysaccharides such as capsularpolysaccharides, LPS, and peptodglycan, and biofilm exopolysaccharide(e.g., alginate, EPS), N-linked glycans, and O-linked glycans. Prebioticsugars may be modified and carbohydrate derivatives include amino sugars(e.g., sialic acid, N-acetylglucosamine, galactosamine), deoxy sugars(e.g., rhamnose, fucose, deoxyribose), sugar phosphates, glycosylamines,sugar alcohols, and acidic sugars (e.g., glucuronic acid, ascorbicacid).

In one embodiment, the prebiotic carbohydrate component of thepharmaceutical composition, dosage form, or kit consists essentially ofone or more non-digestible saccharides. In one embodiment,non-digestible oligosaccharides the non-digestible oligosaccharides aregalactooligosaccharides (GOS). In another embodiment, the non-digestibleoligosaccharides are fructooligosaccharides (FOS).

In one embodiment, the prebiotic carbohydrate component of thepharmaceutical composition, dosage form, or kit allows the commensalcolonic microbiota, comprising microorganisms associated with ahealthy-state microbiome or presenting a low risk of a patientdeveloping an autoimmune or inflammatory condition, to be regularlymaintained. In one embodiment, the prebiotic carbohydrate allows theco-administered or co-formulated microbe or microbes to engraft, grow,and/or be regularly maintained in a mammalian subject. In someembodiments, the mammalian subject is a human subject. In preferredembodiments, the mammalian subject suffers from or is at risk ofdeveloping an autoimmune or inflammatory disorder.

In some embodiments, the prebiotic favors the growth of an administeredmicrobe, wherein the growth of the administered microbe and/or thefermentation of the administered prebiotic by the administered microbeslows or reduces the growth of a pathogen or pathobiont. For example,FOS, neosugar, or inuliri promotes the growth of acid-forming bacteriain the colon such as bacteria belonging to the genera Lactobacillus orBifidobacterium and Lactobacillus acidophilus and Bifidobacteriumbifidus can play a role in reducing the number of pathogenic bacteria inthe colon (see U.S. Pat. No. 8,486,668, PREBIOTIC FORMULATIONS ANDMETHODS OF USE). Other polymers, such as various galactans, lactulose,and carbohydrate based gums, such as psyllium, guar, carrageen, gellan,and konjac, are also known to improve gastrointestinal (GI) health.

In some embodiments, the prebiotic composition of the inventioncomprises one or more of GOS, lactulose, raffinose, stachyose,lactosucrose, FOS (i.e., oligofructose or oligofructan), inulin,isomalto-oligosaccharide, xylo-oligosaccharide, paratinoseoligosaccharide, transgalactosylated oligosaccharides (i.e.,transgalacto-oligosaccharides), transgalactosylate disaccharides,soybean oligosaccharides (i.e., soyoligosaccharides),gentiooligosaccharides, glucooligosaccharides, pecticoligosaccharides,palatinose polycondensates, difructose anhydride III, sorbitol,maltitol, lactitol, polyols, polydextrose, reduced paratinose,cellulose, P3-glucose, 3-galactose, β-fructose, verbascose, galactinol,and β-glucan, guar gum, pectin, high, sodium alginate, and lambdacarrageenan, or mixtures thereof. The GOS may be a short-chain GOS, along-chain GOS, or any combination thereof. The FOS may be a short-chainFOS, a long-chain FOS, or any combination thereof.

In some embodiments, the prebiotic composition comprises twocarbohydrate species (nonlimiting examples being a GOS and FOS) in amixture of at least 1:1, at least 2:1, at least 5:1, at least 9:1, atleast 10:1, about 20:1, or at least 20:1.

In some embodiments, the prebiotic composition of the inventioncomprises a mixture of one or more non-digestible oligosaccharides,non-digestible polysaccharides, free monosaccharides, non-digestiblesaccharides, starch, or non-starch polysaccharides. In one embodiment, aprebiotic component of a prebiotic composition is a GOS composition. Inone embodiment, a prebiotic composition is a pharmaceutical composition.In one embodiment, a pharmaceutical composition is a GOS composition.

Oligosaccharides are generally considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. Most oligosaccharides described herein aredescribed with the name or abbreviation for the non-reducing saccharide(e.g., Gal or D-Gal), preceded or followed by the configuration of theglycosidic bond (α or β), the ring bond, the ring position of thereducing saccharide involved in the bond, and then the name orabbreviation of the reducing saccharide (e.g., Glc or D-Glc). Thelinkage (e.g., glycosidic linkage, galactosidic linkage, glucosidiclinkage) between two sugar units can be expressed, for example, as 1,4,1->4, or (1-4).

Both FOS and GOS are non-digestible saccharides. β glycosidic linkagesof saccharides, such as those found in, but not limited to, FOS and GOS,make these prebiotics mainly non-digestible and unabsorbable in thestomach and small intestine α-linked GOS (α-GOS) is also not hydrolyzedby human salivary amylase, but can be used by Bifidobacterium bifidumand Clostridium butyricum (Yamashita A. et al., 2004. J. Appl. Glycosci.51:115-122). FOS and GOS can pass through the small intestine and intothe large intestine (colon) mostly intact, except where commensalmicrobes and microbes administered as part of a pharmaceuticalcomposition are able to metabolize the oligosaccharides.

GOS (also known as galacto-oligosaccharides, galactooligosaccharides,trans-oligosaccharide (TOS), trans-galacto-oligosaccharide (TGOS), andtrans-galactooligosaccharide) are oligomers or polymers of galactosemolecules ending mainly with a glucose or sometimes ending with agalactose molecule and have varying degree of polymerization (generallythe DP is between 2-20) and type of linkages. In one embodiment, GOScomprises galactose and glucose molecules. In another embodiment, GOScomprises only galactose molecules. In a further embodiment, GOS aregalactose-containing oligosaccharides of the form of[β-D-Gal-(1-6)]_(n)-β-D-Gal-(1-4)-D-Glc wherein n is 2-20. In anotherembodiment, GOS are galactose-containing oligosaccharides of the formGlc α1-4-[β Gal 1-6)]_(n) where n=2-20. In another embodiment, GOS arein the form of α-D-Glc (1-4)-[β-D-Gal-(1-6)-]_(n) where n=2-20. Gal is agalactopyranose unit and Glc (or Glu) is a glucopyranose unit.

In one embodiment, a prebiotic composition comprises a GOS-relatedcompound. A GOS-related compound can have the following properties: a) a“lactose” moiety; e.g., GOS with a gal-glu moiety and any polymerizationvalue or type of linkage; or b) be stimulatory to “lactose fermenting”microbes in the human GI tract; for example, raffinose (gal-fru-glu) isa “related” GOS compound that is stimulatory to both lactobacilli andbifidobacteria.

In one embodiment, a prebiotic composition comprises GOS with a lowdegree of polymerization. In one embodiment a prebiotic compositioncomprising GOS with a low degree of polymerization increases growth ofprobiotic and select commensal bacteria to a greater extent than anequivalent amount of a prebiotic composition comprising GOS with a highdegree of polymerization. In one embodiment, a prebiotic compositioncomprising a high percentage of GOS with a low degree of polymerizationincreases growth of probiotic and beneficial commensal bacteria to agreater extent than an equivalent amount of a prebiotic compositioncomprising a low percentage of GOS with a low degree of polymerization(DP). In one embodiment a prebiotic composition comprises GOS with a DPless than 20, such as less than 10, less than 9, less than 8, less than7, less than 6, less than 5, less than 4, or less than 3. In anotherembodiment a prebiotic composition comprising GOS with a low DPincreases growth of co-formulated or co-administered microbes and/orbeneficial commensal microbes in the GI tract of a subject.

Linkages between the individual sugar units found in GOS and otheroligosaccharides include β-(1-6), β-(1-4), β-(1-3) and β-(1-2) linkages.In one embodiment, the administered oligosaccharides (e.g., GOS) arebranched saccharides. In another embodiment, the administeredoligosacchardies (e.g, GOS) are linear saccharides.

In some embodiments, the GOS comprises a disaccharide Gal α (1-6) Gal,at least one trisaccharide selected from Gal β (1-6)-Gal β (1-4)-Glc andGal β (1-3)-Gal β (1-4)-Glc, the tetrasaccharide Gal β(1-6)-Gal β(1-6)-Gal β (1-4)-Glc and the pentasaccharide Gal β (1-6)-Gal β(1-6)-Gal β (1-6)-Gal β (1-4)-Glc.

In one embodiment, a GOS composition is a mixture of 10 to 45% w/vdisaccharide, 10 to 45% w/v trisaccharide, 10 to 45% w/v tetrasaccharideand 10 to 45% w/v pentasaccharide. In another embodiment, a GOScomposition is a mixture of oligosaccharides comprising 20-28% by weightof β (1-3) linkages, 20-25% by weight of β (1-4) linkages, and 45-55% byweight of β (1-6) linkages. In one embodiment, a GOS composition is amixture of oligosaccharides comprising 26% by weight of β (1-3)linkages, 23% by weight of β (1-4) linkages, and 51% by weight of β(1-6) linkages.

Alpha-GOS (also called alpha-bond GOS or alpha-linked GOS) areoligosaccharides having an alpha-galactopyranosyl group. Alpha-GOScomprises at least one alpha glycosidic linkage between the saccharideunits. Alpha-GOS are generally represented by α-(Gal)_(n) (n usuallyrepresents an integer of 2 to 10) or α-(Gal)_(n) Glc (n usuallyrepresents an integer of 1 to 9). Examples include a mixture ofα-galactosylglucose, α-galactobiose, α-galactotriose, α-galactotetraose,and higher oligosaccharides. Additional non-limiting examples includemelibiose, manninootriose, raffinose, stachyose, and the like, which canbe produced from beat, soybean oligosaccharide, and the like.

Commercially available and enzyme synthesized alpha-GOS products arealso useful for the compositions described herein. Synthesis ofalpha-GOS with an enzyme is conducted utilizing the dehydrationcondensation reaction of α-galactosidase with the use of galactose,galactose-containing substance, or glucose as a substrate. Thegalactose-containing substance includes hydrolysates ofgalactose-containing substances, for example, a mixture of galactose andglucose obtained by allowing beta-galactosidase to act on lactose, andthe like. Glucose can be mixed separately with galactose and be used asa substrate with α-galactosidase (see e.g., WO 02/18614). Methods ofpreparing alpha-GOS have been described (see e.g., EPI 514551 andEP2027863).

In one embodiment, a GOS composition comprises a mixture of saccharidesthat are alpha-GOS and saccharides that are produced bytransgalactosylation using P3-galactosidase. In another embodiment, GOScomprises alpha-GOS. In another embodiment, alpha-GOS comprises α-(Gal)₂from 10% to 100% by weight. In one embodiment, GOS comprises onlysaccharides that are produced by transgalactosylation usingβ-galactosidase.

In one embodiment, a GOS composition can comprise GOS with alphalinkages and beta linkages.

In one embodiment, the pharmaceutical composition, dosage form, or kitcomprises, in addition to one or more microbes, an oligosaccharidecomposition that is a mixture of oligosaccharides comprising 1-20% byweight of di-saccharides, 1-20% by weight tri-saccharides, 1-20% byweight tetra-saccharides, and 1-20% by weight penta-saccharides. Inanother embodiment, an oligosaccharide composition is a mixture ofoligosaccharides consisting essentially of 1-20% by weight ofdi-saccharides, 1-20% by weight tri-saccharides, 1-20% by weighttetra-saccharides, and 1-20% by weight penta-saccharides.

In one embodiment, a prebiotic composition is a mixture ofoligosaccharides comprising 1-20% by weight of saccharides with a degreeof polymerization (DP) of 1-3, 1-20% by weight of saccharides with DP of4-6, 1-20% by weight of saccharides with DP of 7-9, and 1-20% by weightof saccharides with DP of 10-12, 1-20% by weight of saccharides with DPof 13-15.

In another embodiment, a prebiotic composition comprises a mixture ofoligosaccharides comprising 50-55% by weight of di-saccharides, 20-30%by weight tri-saccharides, 10-20% by weight tetra-saccharide, and 1-10%by weight penta-saccharides. In one embodiment, a GOS composition is amixture of oligosaccharides comprising 52% by weight of di-saccharides,26% by weight tri-saccharides, 14% by weight tetra-saccharide, and 5% byweight penta-saccharides. In another embodiment, a prebiotic compositioncomprises a mixture of oligosaccharides comprising 45-55% by weighttri-saccharides, 15-25% by weight tetra-saccharides, 1-10% by weightpenta-saccharides.

In certain embodiments, the composition according to the inventioncomprises a mixture of neutral and acid oligosaccharides as disclosed inWO 2005/039597 (N.V. Nutricia) and US Patent Application 20150004130,which are hereby incorporated by reference. In one embodiment, the acidoligosaccharide has a degree of polymerization (DP) between 1 and 5000.In another embodiment, the DP is between 1 and 1000. In anotherembodiment, the DP is between 2 and 250. If a mixture of acidoligosaccharides with different degrees of polymerization is used, theaverage DP of the acid oligosaccharide mixture is preferably between 2and 1000. The acid oligosaccharide may be a homogeneous or heterogeneouscarbohydrate. The acid oligosaccharides may be prepared from pectin,pectate, alginate, chondroitine, hyaluronic acids, heparin, heparane,bacterial carbohydrates, sialoglycans, fucoidan, fucooligosaccharides orcarrageenan, and are preferably prepared from pectin or alginate. Theacid oligosaccharides may be prepared by the methods described in WO01/60378, which is hereby incorporated by reference. The acidoligosaccharide is preferably prepared from high methoxylated pectin,which is characterized by a degree of methoxylation above 50%. As usedherein, “degree of methoxylation” (also referred to as DE or “degree ofesterification”) is intended to mean the extent to which free carboxylicacid groups contained in the polygalacturonic acid chain have beenesterified (e.g. by methylation). In some embodiments, the acidoligosaccharides have a degree of methoxylation above about 10%, aboveabout 20%, above about 50%, above about 70%. In some embodiments, theacid oligosaccharides have a degree of methylation above about 10%,above about 20%, above about 50%, above about 70%.

The term neutral oligosaccharides as used in the present inventionrefers to saccharides which have a degree of polymerization of monoseunits exceeding 2, exceeding 3, exceeding 4, or exceeding 10, which arenot or only partially digested in the intestine by the action of acidsor digestive enzymes present in the human upper digestive tract (smallintestine and stomach) but which are fermented by the human intestinalflora and preferably lack acidic groups. The neutral oligosaccharide isstructurally (chemically) different from the acid oligosaccharide. Theterm neutral oligosaccharides as used herein preferably refers tosaccharides which have a degree of polymerization of the oligosaccharidebelow 60 monose units. The term monose units refers to units having aclosed ring structure e.g., the pyranose or furanose forms. In comeembodiments, the neutral oligosaccharide comprises at least 90% or atleast 95% monose units selected from the group consisting of mannose,arabinose, fructose, fucose, rhamnose, galactose, -D-galactopyranose,ribose, glucose, xylose and derivatives thereof, calculated on the totalnumber of monose units contained therein. Suitable neutraloligosaccharides are preferably fermented by the gut flora. Nonlimitingexamples of suitable neutral oligosaccharides are cellobiose(4-O-β-D-glucopyranosyl-D-glucose), cellodextrins((4-O-β-D-glucopyranosyl)n-D-glucose), B-cyclo-dextrins (Cyclicmolecules of α-1-4-linked D-glucose; α-cyclodextrin-hexamer,β-cyclodextrin-heptamer and γ-cyclodextrin-octamer), indigestibledextrin, gentiooligosaccharides (mixture of β-1-6 linked glucoseresidues, some 1-4 linkages), glucooligosaccharides (mixture ofα-D-glucose), isomaltooligosaccharides (linear α-1-6 linked glucoseresidues with some 1-4 linkages), isomaltose(6-O-α-D-glucopyranosyl-D-glucose); isomaltriose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-D-glucose), panose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-(1-4)-D-glucose),leucrose (5-O-α-D-glucopyranosyl-D-fructopyranoside), palatinose orisomaltulose (6-O-α-D-glucopyranosyl-D-fructose), theanderose(O-α-D-glucopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-B-D-fructofuranoside), D-agatose, D-lyxo-hexylose, lactosucrose(O-β-D-galactopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside),α-galactooligosaccharides including raffinose, stachyose and other soyoligosaccharides(O-α-D-galactopyranosyl-(1-6)-α-D-glucopyranosyl-(β-D-fructofuranoside),β-galactooligosaccharides or transgalacto-oligosaccharides(β-D-galactopyranosyl-(1-6)-[β-D-glucopyranosyl]n-(1-4) α-D glucose),lactulose (4-O-β-D-galactopyranosyl-D-fructose), 4′-galatosyllactose(O-D-galactopyranosyl-(1-4)-O-β-D-glucopyranosyl-(1-4)-D-glucopyranose),synthetic galactooligosaccharide (neogalactobiose, isogalactobiose,galsucrose, isolactose I, II and III), fructans-Levan-type(β-D-(2→6)-fructofuranosyl)n α-D-glucopyranoside), fructans-Inulin-type(β-D-((2→1)-fructofuranosyl)n α-D-glucopyranoside), 1f-β-fructofuranosylnystose (β-D-((2→1)-fructofuranosyl)nB-D-fructofuranoside), xylooligo-saccharides (B-D-((1→4)-xylose)n,lafinose, lactosucrose and arabinooligo saccharides.

In some embodiments, the neutral oligosaccharide is selected from thegroup consisting of fructans, fructooligosaccharides, indigestibledextrins galactooligo-saccharides (includingtransgalactooligosaccharides), xylooligosaccharides,arabinooligo-saccharides, glucooligosaccharides, mannooligosaccharides,fucooligosaccharides and mixtures thereof.

Suitable oligosaccharides and their production methods are furtherdescribed in Laere K. J. M. (Laere, K. J. M., Degradation ofstructurally different non-digestible oligosaccharides by intestinalbacteria: glycosylhydrolases of Bi. adolescentis. PhD-thesis (2000),Wageningen Agricultural University, Wageningen, The Netherlands), theentire content of which is hereby incorporated by reference.Transgalactooligosaccharides (TOS) are for example sold under thetrademark Vivinal™ (Borculo Domo Ingredients, Netherlands). Indigestibledextrin, which may be produced by pyrolysis of corn starch, comprisesα(1→4) and α(1→6) glucosidic bonds, as are present in the native starch,and contains 1→2 and 1→3 linkages and levoglucosan. Due to thesestructural characteristics, indigestible dextrin containswell-developed, branched particles that are partially hydrolysed byhuman digestive enzymes. Numerous other commercial sources ofindigestible oligosaccharides are readily available and known to skilledpersons in the art. For example, transgalactooligosaccharide isavailable from Yakult Honsha Co., Tokyo, Japan. Soybean oligosaccharideis available from Calpis Corporation distributed by Ajinomoto U.S.A.Inc., Teaneck, N.J.

In a further preferred embodiment, the prebiotic mixture of thepharmaceutical composition described herein comprises an acidoligosaccharide with a DP between 1 and 5000, prepared from pectin,alginate, and mixtures thereof; and a neutral oligosaccharide, selectedfrom the group of fructans, fructooligosaccharides, indigestibledextrins, galactooligosaccharides includingtransgalacto-oligosaccharides, xylooligosaccharides,arabinooligosaccharides, glucooligosaccharides, manno-oligosaccharides,fucooligosaccharides, and mixtures thereof.

In certain embodiments, the prebiotic mixture comprises xylose. In otherembodiments, the prebiotic mixture comprises a xylose polymer (i.e.xylan). In some embodiments, the prebiotic comprises xylose derivatives,such as xylitol, a sugar alcohol generated by reduction of xylose bycatalytic hydrogenation of xylose, and also xylose oligomers (e.g.,xylooligosaccharide). While xylose can be digested by humans, viaxylosyltransferase activity, most xylose ingested by humans is excretedin urine. In contrast, some microorganisms are efficient at xylosemetabolism or may be selected for enhanced xylose metabolism. Microbialxylose metabolism may occur by at least four pathways, including theisomerase pathway, the Weimburg pathway, the Dahms pathway, and, foreukaryotic microorganisms, the oxido-reductase pathway.

The xylose isomerase pathway involves the direct conversion of D-xyloseinto D-xylulose by xylose isomerase, after which D-xylulose isphosphorylated by xylulose kinase to yield D-xylolose-5-phosphate, anintermediate of the pentose phosphate pathway.

In the Weimberg pathway, D-xylose is oxidized to D-xylono-lactone by aD-xylose dehydrogenase. Then D-xylose dehydrogenase is hydrolyzed by alactonase to yield D-xylonic acid, and xylonate dehydratase acitivitythen yields 2-keto-3-deoxy-xylonate. The final steps of the Weimbergpathway are a dehydratase reaction to form 2-keto glutarate semialdehydeand an oxidizing reaction to form 2-ketoglutarate, an intermediate ofthe Krebs cycle.

The Dahms pathway follows the same mechanism as the Weimberg pathway butdiverges once it has yielded 2-keto-3-deoxy-xylonate. In the Dahmspathway, an aldolase splits 2-keto-β-deoxy-xylonate into pyruvate andglycolaldehyde.

The xylose oxido-reductase pathway, also known as the xylosereductase-xylitol dehydrogenase pathway, begins by the reduction ofD-xylose to xylitol by xylose reductase followed by the oxidation ofxylitol to D-xylulose by xylitol dehydrogenase. As in the isomerasepathway, the next step in the oxido-reductase pathway is thephosphorylation of D-xylulose by xylulose kinase to yieldD-xylolose-5-phosphate.

Xylose is present in foods like fruits and vegetables and other plantssuch as trees for wood and pulp production. Thus, xylose can be obtainedin the extracts of such plants. Xylose can be obtained from variousplant sources using known processes including acid hydrolysis followedby various types of chromatography. Examples of such methods to producexylose include those described in Maurelli, L. et al. (2013), Appl.Biochem. Biotechnol. 170:1104-1118; Hooi H. T et al. (2013), Appl.Biochem. Biotechnol. 170:1602-1613; Zhang H-J. et al. (2014), BioprocessBiosyst. Eng. 37:2425-2436.

Preferably, the metabolism of xylose and/or the shift in microbiota dueto the metabolism of the xylose provided in a pharmaceutical compositionof the invention confers a benefit to a host, e.g. immunologicaltolerance. For example, in aspects in which the patient is at risk orsuffering from GVHD, the immunological tolerance may reducegraft-versus-host activity while maintaining graft-versus-leukemiaactivity. The xylose may be, e.g. i) cytotoxic for an autoimmunedisease- and/or inflammatory disease-associated associated pathogen orpathobiont, ii) cytostatic for an autoimmune disease- and/orinflammatory disease-associated pathogen or pathobiont, iii) capable ofdecreasing the growth of autoimmune disease- and/or inflammatorydisease-associated pathogen or pathobiont, iv) capable of inhibiting thegrowth of an autoimmune disease- and/or inflammatory disease-associatedpathogen or pathobiont, v) capable of decreasing the colonization of anautoimmune disease- and/or inflammatory disease-associated pathogen orpathobiont, vi) capable of inhibiting the colonization of an autoimmunedisease- and/or inflammatory disease-associated pathogen or pathobiont,vii) capable of eliciting an immunomodulatory response in the host thatreduces the risk of an autoimmune and/or inflammatory disorder, viii),capable of eliciting an immunomodulatory response in the host thatreduces the severity of an autoimmune and/or inflammatory disorder, ix)capable of promoting barrier integrity directly or indirectly throughits impact on microbiota, or x) any combination of i)-ix).

In some embodiments, the pharmaceutical composition or dosage formcomprises a bacterial population and xylose in an amount effective topromote the growth of select bacteria of the family Clostridiacea,including members of the genus Clostridium, Ruminococcus, or Blautia orrelatives thereof in a host. In some embodiments, the pharmaceuticalcomposition or dosage form is further effective to promote theproliferation of select bacteria of the family Clostridiacea, includingmembers of the genus Clostridium, Ruminococcus, or Blautia or relativesthereof in a host. In certain embodiments, the pharmaceuticalcomposition or dosage form comprises a bacterial population and xylosein an amount effective to promote the colonization and/or engraftment ofselect bacteria of the family Clostridiacea, including members of thegenus Clostridium, Ruminococcus, or Blautia or relatives thereof in ahost. In preferred embodiments, the pharmaceutical composition or dosageform is further capable of altering a dysbiotic state such that thegrowth, proliferation, colonization, and/or engraftment of a host by apathogen, pathobiont, disease-associated microbe, or a combinationthereof such that the population of at least one pathogen, pathobiont,or disease-associated microbe is decreased 2-fold, 5-fold, 10-fold,50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 10000-fold, or over10000-fold. In one embodiment, the pharmaceutical composition or dosageform is capable of locally or systemically eliminating at least onepathogen, pathobiont, or disease-associated microbe from a host.

In some embodiments, the prebiotic mixture comprises a carbohydratemonomer or polymer that has been modified i.e., substituted with othersubstituents (e.g., acetyl group, glucuronic acid residue, arabinoseresidue, or the like) (see US Patent Application 20090148573, herebyincorporated by reference). The term “modified”, as used herein, refersto a molecule modified from a reference molecule, and includes not onlyartificially produced molecules but also naturally occurring molecules.In preferred embodiments, the modification occurs at one or morehydroxyl groups of the reference carbohydrate. In some embodiments, themodification occurs at carbon-2 (C2), the modification occurs atcarbon-6 (C6), or a combination thereof.

In some embodiments, a carbohydrate (α monomer or, preferably, apolymer) is modified with one or more hydrophilic groups. Nonlimitingexamples of the hydrophilic groups include an acetyl group, a4-O-methyl-α-D-glucuronic acid residue, an L-arabinofuranose residue, anL-arabinose residue, and an α-D-glucuronic acid residue. In someembodiments, the modification is the replacement of one or more hydroxylgroups with —H, —CH₂OH, —CH₃, or —NH₂.

In some embodiments, the composition comprises at least one carbohydratethat elicits an immunomodulatory response. Exemplary immunomodularycarbohydrates include (but are not limited to) fructo-oligosaccharides,glycosaminoglycans (e.g., heparin sulfate, chondroitin sulfate A,hyaluronan), O-glycans, and carrageenan oligosaccharides, andgalacto-oligosaccharides. Immunomodulatory carbohydrates may be purifiedfrom plants or microbes or may be synthetically derived.Immunomodulatory carbohydrates may be effective to, for example, preventdisease, suppress symptoms, treat disease, or any combination thereof.

In some embodiments, immunomodulatory carbohydrates are C-type lectinreceptor ligands. In preferred embodiments, the C-type lectin receptorligands are produced by one or more fungal species. In otherembodiments, the immunomodulatory carbohydrates are bacterialexopolysaccharides, such as (but not limited to) the exopolysaccharides(EPS) produced by Bacillus subtilis, Bifidobacterium breve, orBacteroides fragilis. In some aspects, immunomodulatory carbohydratesare zwitterionic polysaccharides. In some aspects, immunomodulatorycarbohydrates modulate toll-like receptor 2 (TLR2) and/or toll-likereceptor 4 (TLR4) responses in a host. For example, autoimmune orinflammatory diseases characterized by intestinal inflammation may beprevented by a TLR4 agonist such as but not limited to B. subtilis EPS(Jones S, Paynich M L, Kearns D B, Knight K L, 2014. Protection fromIntestinal Inflammation by Bacterial Exopolysaccharides. The Journal ofImmunology. 192:4813-4820). Immunomodulatory carbohydrates may alsoactivate CD4+ T cells and/or lead to an upregulation of theanti-inflammatory cytokine interleukin-10 (Mazmanian S K, Kasper D L,2006. The love-hate relationship between bacterial polysaccharides andthe host immune system. Nat. Rev. Immunol. 6: 849-858). Immunomodulatorycarbohydrates may be selected for administration to a patient based onthe presence, abundance, distribution, modification and/or linkages ofsugar residues. For example, immunomodulatory carbohydrates used in theprevention of intestinal disorders or autoimmune conditions thatmanifest in the gut (non-limiting examples being IBD and GVHD) may beselected based on i) a high abundance of mannose residues; ii) thepresence of terminal mannopyransosyl (t-Man) residues and/or 2,6 linkedmannopyranosyl residues (2,6-Man), iii) a ratio of mannose to glucoseresidues in the approximate range of 8:2 to 9:1, iv) the presence ofgalactose residues, v) areas of positive charge, or vi) a combinationthereof.

Carbohydrates may be selected according to the fermentation or metabolicpreferences of a microbe selected for administration to a mammaliansubject. Selection criteria include but are not limited to sugarcomplexity (e.g., monosaccharides, including but not limited to glucose,versus oligosaccharides or starches) as well as by desired end-product.Non-limiting examples include the fermentation products ethanol andcarbon dioxide (CO₂) (e.g., via ethanol fermentation by Saccharomycessp. Zymomonas sp.), lactate (e.g., via homolactic acid fermentation byLactococcus sp., Streptococcus sp., Enterococcus sp., Pediococcus sp.and some species Lactobacillus), lactate, ethanol, and CO₂ (e.g., viaheterolactic acid fermentation (which includes the phosphoketolasepathway) by some species of Lactobacillus as well as Leuconostoc sp.,Oenococcus sp., and Weissella sp.), butanol, acetone, CO₂ and H₂ (viaacetone-butanol fermentation by some Clostridium sp.), and short chainfatty acids (with or without the production of other products) (MullerV, 2011. Bacterial Fermentation. Encyclopedia of Life Sciences).Examples of fermentation leading to short chain fatty acid productioninclude homoacetic acid fermentation (e.g., by Acetobacterium sp., andresulting in acetate), propionic acid fermentation (e.g., byPropionibacterium sp., and resulting in propionate, acetate and CO₂)mixed acid fermentation (e.g., by Escherichia sp., and resulting inethanol, lactate, acetate, succinate, formate, CO₂, and H₂), butyratefermentation (e.g., by some Clostridium sp., resulting in butyrate, CO₂,and H₂), and 2,3-butanediol fermentation (e.g., by Enterobacter sp.,resulting in ethanol, butanediol, lactate, formate, CO₂, and H₂). Insome embodiments, selection of carbohydrates for co-formulation ofco-administration with a type of microbe or types of microbe may beachieved by computational analysis of microbial enzymatic pathways,including but not limited to the presence of metabolic/fermentationpathway enzymes including but not limited to the enzymes provided inTable 4.

Other prebiotics include molecules capable of selective orsemi-selective utilization by microbes of the composition containedherein. The ability of a microbe to utilize a metabolite of interest isdetermined by the genomic capacity of that microbe. Public databaseshave characterized many microbes and automate the annotation of thegenome to allow a computational analysis of the metabolites a microbe ispotentially able to utilize. Databases such as the Cluster ofOrthologous Groups (COGs) database characterize genomes from a varietyof species in this manner and are capable of characterizing newlysequenced genomes as well (e.g. see in this fashion (Tatusov et al 2000.Nucl Acid Res). Furthermore, pathway analysis classifies COGs intodifferent categories with associated one letter codes including J,translation; L replication, recombination, and repair, K transcription;O molecular chaperones and related functions, M, cell wall structure andbiogenesis and outer membrane, N secretion motility and chemotaxis; Tsignal Transduction; P inorganic ion transport and metabolism; C energyproduction and conversion; G, carbohydrate metabolism and transport; Eamino acid metabolism and transport; F, nueclotide metabolism andtransport; D cell Division and chromosome partitioning; R generalfunctional prediction. In preferred embodiments, COGs of the categories,N, M, P, C, G, E, and F are selected as preferred COGs to both provideenhanced growth on specific substrates and modified behaviors relevantfor anti-tumor properties. Other preferred embodiments, include COGs forC, G, E, and specific COG functions are listed in Table 4.

COGs are selected to be specific or semi enriched in the host or othermicrobes within a host by searching for specific functions present inthe microbe of interest but absent from a large set of other competitionorganisms. Tissue specific analysis of the host for enzymes expressedwithin a tissue is performed to identify tissue specific enzymaticactivities in the host. Specific functions are absent from at least 90%,at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, atleast 30% at least 20% or at least 10% of the other organisms selectedfrom the group of the host, the host tissue, the disease-associatedmicrobiota, the host gut microbiota, the host niche specific to theengraftment of the microbial composition (e.g. GI tract, skin).

Once these COGs are identified, databases like KEGG were used to linkthe enzymatic functions to identify the metabolites that are substratesfor these selective COGs. Furthermore, the selective analysis togenerate selective metabolites is repeated on the set of substrate ofCOGs to validate that the pathways and metabolites are selective to thedesired microbial composition.

Also provided are co-formulations of microbial populations andcarbohydrates or other materials that foster desired microbial growthwhile, optionally, inhibiting undesired microbial growth. For example,one or more bacterial entities are encapsulated in a carbohydrate layeror coating (exemplary formulations include xylose-PEG and orxylose-PEG-PLGA).

Selecting Prebiotics for Particular Probiotics

It is well known that organisms, including bacteria, show a preferentialand hierarchical utilization of different carbohydrates. Some bacteriawill not respond at all to a sugar, while some bacterial will use asugar preferentially. The metabolic effects of a sugar on a bacteriareflect how the bacteria senses and responds to its environment.Providing a sugar to a bacteria that has preferential utiliziation canencourage its growth/selection. Conversely, providing a sugar to abacteria that is not preferred may lead to its down selection. Forexample, a particular sugar may not be a preferred substrate formetabolism, and thus may be utilized to bias for or enhance the growthand/or proliferation of particular microbial (e.g., bacterial) speciesor strains. Further, a particular sugar or the metabolism thereof mayact as a selector to promote the survival, colonization, and/orengraftment of a desired microbial population in a host. Alternativelyor simultaneously, a particular sugar or the metabolism thereof may actas a selector to reduce or eliminate the survival, colonization, and/orengraftment of an undesired microbial population in host.

Carbohydrates may be selected according to the fermentation or metabolicpreferences of a microbe selected for administration to a mammaliansubject. Selection criteria include but are not limited to sugarcomplexity (e.g., monosaccharides, including but not limited to glucose,versus oligosaccharides or starches) as well as by desired end-product.Non-limiting examples include the fermentation products ethanol andcarbon dioxide (CO₂) (e.g., via ethanol fermentation by Saccharomycessp. Zymomonas sp.), lactate (e.g., via homolactic acid fermentation byLactococcus sp., Streptococcus sp., Enterococcus sp., Pediococcus sp.and some species Lactobacillus), lactate, ethanol, and CO₂ (e.g., viaheterolactic acid fermentation (which includes the phosphoketolasepathway) by some species of Lactobacillus as well as Leuconostoc sp.,Oenococcus sp., and Weissella sp.), butanol, acetone, CO₂ and H₂ (viaacetone-butanol fermentation by some Clostridium sp.), and short chainfatty acids (with or without the production of other products) (MullerV, 2011. Bacterial Fermentation. Encyclopedia of Life Sciences).Examples of fermentation leading to short chain fatty acid productioninclude homoacetic acid fermentation (e.g., by Acetobacterium sp., andresulting in acetate), propionic acid fermentation (e.g., byPropionibacterium sp., and resulting in propionate, acetate and CO₂)mixed acid fermentation (e.g., by Escherichia sp., and resulting inethanol, lactate, acetate, succinate, formate, CO₂, and H₂), butyratefermentation (e.g., by some Clostridium sp., resulting in butyrate, CO₂,and H₂), and 2,3-butanediol fermentation (e.g., by Enterobacter sp.,resulting in ethanol, butanediol, lactate, formate, CO₂, and H₂). Insome embodiments, selection of carbohydrates for co-formulation orco-administration with a type of microbe or types of microbe may beachieved by computational analysis of microbial enzymatic pathways,including but not limited to the presence of metabolic/fermentationpathway enzymes including but not limited to the enzymes provided inTable 4.

In preferred embodiments, the combination of a type of microbe ormicrobial composition and type of prebiotic mixture is selected based onthe fermentation or metabolic preferences of one or more microbescapable of producing immunomodulatory SCFAs (e.g., preference forcomplex versus simple sugar or preference for a fermentation productversus a prebiotic). For example, M. eldsenii prefers lactatefermentation to glucose fermentation, and maximization of propionateproduction by M. eldsenii in a mammalian subject may therefore beachieved by administering along with M. eldsenii a favored substrate(e.g., lactate) or one or more microbes capable of fermenting glucoseinto lactate (e.g., Streptococcus bovis) (Hosseini E., et al. 2011.Propionate as a health-promoting microbial metabolite in the human gut.Nutrition Reviews. 69(5): 245-258).

Immunomodulation can also be achieved by the microbial production ofglutathione or gamma-glutamylcysteine. Thus, in certain embodiments, thepharmaceutical composition, dosage form, or kit comprises at least onetype of microbe capable of producing glutathione and/orgamma-glutamylcysteine

In some aspects, the composition, dosage form, or kit comprises one ormore microbes selected for the presence of glutamate cysteine ligase(e.g., Lactobacillus fermentum) and/or L-proline biosynthesis enzymes(e.g., E. coli) (Peran et al., 2006. Lactobacillus fermenum, a probioticcapable to release glutathione, prevents colonic inflammation in theTNBS model of rat colitis. Int J Colorectal Dis. 21(8): 737-746;Veeravalli et al., 2011. Laboratory evolution of glutathionebiosynthesis reveals naturally compensatory pathways. Nat Chem Bio.7(2): 101-105). In a preferred embodiment, at least one microbe in thepharmaceutical composition, dosage form, or kit is L. fermentum.

VII. Methods of Altering the Microbiome Using Prebiotics and/orProbiotics

Disclosed herein are therapeutic compositions containing non-pathogenic,germination-competent bacterial entities and/or fungal entities, for theprevention, control, and treatment of immune and inflammatory diseases,disorders and conditions and for general nutritional health. Thesecompositions are advantageous in being suitable for safe administrationto humans and other mammalian subjects and are efficacious in numerousimmune and inflammatory diseases, disorders and conditions and ingeneral nutritional health. While spore-based compositions are known,these are generally prepared according to various techniques such aslyophilization or spray-drying of liquid bacterial cultures, resultingin poor efficacy, instability, substantial variability and lack ofadequate safety.

It has now been found that populations of bacterial entities and/orfungal entities can be obtained from biological materials obtained frommammalian subjects, including humans. These populations are formulatedinto compositions as provided herein, and administered to mammaliansubjects using the methods as provided herein.

Purified Spore Populations.

In some embodiments, the bacterial compositions comprise purified sporepopulations. As described herein, purified spore populations containcombinations of commensal bacteria of the human gut microbiota with thecapacity to meaningfully provide functions of a healthy microbiota whenadministered to a mammalian subject. Without being limited to a specificmechanism, it is thought that such compositions inhibit the growth of apathogen such as C. difficile, Salmonella spp., enteropathogenic E.coli, Fusobacterium spp., Klebsiella spp. and vancomycin-resistantEnterococcus spp., so that a healthy, diverse and protective microbiotacan be maintained or, in the case of pathogenic bacterial infections,repopulate the intestinal lumen to reestablish ecological control overpotential pathogens. In one embodiment, the purified spore populationscan engraft in the host and remain present for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 25 days, 30days, 60 days, 90 days, or longer than 90 days. Additionally, thepurified spore populations can induce other healthy commensal bacteriafound in a healthy gut to engraft in the host that are not present inthe purified spore populations or present at lesser levels and thereforethese species are considered to “augment” the delivered sporepopulations. In this manner, commensal species augmentation of thepurified spore population in the recipient's gut leads to a more diversepopulation of gut microbiota then present initially.

Preferably, the one or more microbes provided in a therapeuticcomposition act additively, more preferably synergistically to confer abenefit to a host, e.g. immunological tolerance. For example, in aspectsin which the patient is at risk or suffering from GVHD, theimmunological tolerance may reduce graft-versus-host activity whilemaintaining graft-versus-leukemia activity. In another example, inaspects in which the patient suffers from Celiac disease, theimmunological tolerance prevents an inappropriate immune response togluten. The microbes may additively or synergistically be, e.g. i)cytotoxic for an autoimmune disease- and/or inflammatorydisease-associated associated pathogen or pathobiont, ii) cytostatic foran autoimmune disease- and/or inflammatory disease-associated pathogenor pathobiont, iii) capable of decreasing the growth of autoimmunedisease- and/or inflammatory disease-associated pathogen or pathobiont,iv) capable of inhibiting the growth of an autoimmune disease- and/orinflammatory disease-associated pathogen or pathobiont, v) capable ofdecreasing the colonization of an autoimmune disease- and/orinflammatory disease-associated pathogen or pathobiont, vi) capable ofinhibiting the colonization of an autoimmune disease- and/orinflammatory disease-associated pathogen or pathobiont, vii) capable ofeliciting an immunomodulatory response in the host that reduces the riskof an autoimmune and/or inflammatory disorder, viii), capable ofeliciting an immunomodulatory response in the host that reduces theseverity of an autoimmue and/or inflammatory disorder, or ix) anycombination of i)-viii).

The microbes described herein may additively or synergistically reducethe number of types of autoimmune disease- or inflammatorydisease-associated pathogens or pathobionts either distally—e.g.,orally-administered microbes reduce the total microbial burden in anorgan not in the gastrointestinal tract, or intravaginally-administeredmicrobes reduce the total microbial burden in an organ that is not thevagina—or locally, e.g., the intestines or vagina, respectively. Distalsites include but are not limited to the liver, spleen, fallopian tubesand uterus.

Thus provided are compositions formulated for vaginal administration,such as bacterial populations. The bacterial populations are capable oftranslocating across vaginal tissue to distal sites, or relocation fromthe vaginal canal into the gastrointestinal tract.

Similarly, the microbes described herein may additively orsynergistically elicit an immunomodulatory response either distally,e.g., in which enteral administration of microbes results in alteringthe immune response at the skin or liver, or locally, e.g. the enteraladministration of microbes results in altering the immune response inthe intestines.

In some situations, the recipient subject is immunocompromised orimmunosuppressed, or is at risk of developing an immune or inflammatorydisorder.

Methods for Administrating Bacterial Compositions to Treat a Subject.

Administration of Microbial Compositions, with or without Prebiotics.

The microbial compositions of the invention, with or without one or moreprebiotics, are suitable for administration to mammals and non-mammaliananimals in need thereof. In certain embodiments, the mammalian subjectis a human subject who has one or more symptoms of a dysbiosis,including but not limited to overgrowth of an undesired pathobiont orpathogen, reduced representation of key bacterial taxa such as theBacteroidetes or Firmicutes or genera or species thereof, or reduceddiversity of microbial species compared to a healthy individual, orreduced overall abundance of anaerobic bacteria.

When the mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms. In some embodiments, the composition isadministered following a successful course of antibiotics to preventdysbiosis and enhance recovery of a diverse, healthy microbiota.

In some embodiments, the disease, disorder or condition characterized byan aberrant microbiota is GVHD.

In some embodiments, the therapeutic composition is administered onlyonce prior to improvement of the disease, disorder or condition. In someembodiments the therapeutic composition is administered at intervalsgreater than two days, such as once every three, four, five or six days,or every week or less frequently than every week. Or the preparation maybe administered intermittently according to a set schedule, e.g., once aday, once weekly, or once monthly, or when the subject relapses from theprimary illness. In another embodiment, the preparation may beadministered on a long-term basis to individuals who are at risk forinfection with or who may be carriers of these pathogens, includingindividuals who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other healthcare professionals).

In embodiments where a subject is administered a probiotic compositionand a prebiotic composition, the probiotic and prebiotic can beadministered simultaneously. For example, the probiotic composition cancontain a prebiotic, or can be administered at the same time as aprebiotic. In other embodiments, the probiotic and the prebiotic aredosed on different regimens. For example, the prebiotic can be dosedprior to or after administration of the probiotic. In other embodiments,the prebiotic can be dosed regularly, and the probiotic is dosed atintervals of reduced frequency compared to dosing of the prebiotic.

Also provided are methods of treating or preventing a mammalian subjectsuffering from or at risk of developing a metabolic disease, anddisorder or condition selected from the group consisting of diabetes,metabolic syndrome, obesity, heart disease, autoimmune disease, liverdisease, and autism using the therapeutic compositions provided herein.

In embodiments, the microbial composition is administered enterically,with or without prebiotics. This preferentially includes oraladministration, or by an oral or nasal tube (including nasogastric,nasojejunal, oral gastric, or oral jejunal). In other embodiments,administration includes rectal administration (including enema,suppository, or colonoscopy). The microbial composition may beadministered to at least one region of the gastrointestinal tract,including the mouth, esophagus, stomach, small intestine, largeintestine, and rectum. In some embodiments, it is administered to allregions of the gastrointestinal tract. The microbial compositions may beadministered orally in the form of medicaments such as powders,capsules, tablets, gels or liquids. The microbial compositions may alsobe administered in gel or liquid form by the oral route or through anasogastric tube, or by the rectal route in a gel or liquid form, byenema or instillation through a colonoscope or by a suppository. In someembodiments, the microbial composition of the above invention isadministered enterically with one ore more prebiotics.

If the composition is administered colonoscopically and, optionally, ifthe microbial composition, with or without one or more prebiotics, isadministered by other rectal routes (such as an enema or suppository) oreven if the subject has an oral administration, the subject may have acolonic-cleansing preparation. The colon-cleansing preparation canfacilitate proper use of the colonoscope or other administrationdevices, but even when it does not serve a mechanical purpose it canalso maximize the proportion of the bacterial composition relative tothe other organisms previously residing in the gastrointestinal tract ofthe subject. Any ordinarily acceptable colonic-cleansing preparation maybe used such as those typically provided when a subject undergoes acolonoscopy.

To evaluate the subject, symptoms of dysbiosis are evaluated posttreatment ranging from 1 day to 6 months after administration of thepurified bacterial population. Fecal material is collected during thisperiod and the microbes present in the gastrointestinal tract can beassessed by 16S rDNA or metagenomic sequencing analysis or otheranalyses commonly used by the skilled artisan. Repopulation by speciesprovided by the spore population as well as Augmentation by commensalmicrobes not present in the spore population will occur in this time asthe spore population catalyzes a reshaping of the gut or vagina ecologyto a state of healthy biosis.

Methods of Treating a Subject.

In some embodiments, the compositions disclosed herein are administeredto a patient or a user (sometimes collectively referred to as a“subject”). As used herein “administer” and “administration” encompassesembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose, and alsosituations in which a user uses a bacteria composition in a certainmanner and/or for a certain purpose independently of or in variance toany instructions received from a second person. Non-limiting examples ofembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose includewhen a physician prescribes a course of conduct and/or treatment to apatient, when a parent commands a minor user (such as a child) toconsume a bacterial composition, when a trainer advises a user (such asan athlete) to follow a particular course of conduct and/or treatment,and when a manufacturer, distributer, or marketer recommends conditionsof use to an end user, for example through advertisements or labeling onpackaging or on other materials provided in association with the sale ormarketing of a product.

The microbial compositions, with or without one or more prebiotics,offer a protective and/or therapeutic effect against GVHD. In someembodiments, the compositions are administered to a subject before thesubject receives a transplant. In other embodiments, the compositionsare administered to a subject concurrently with receiving a transplant.In other embodiments, the compositions are administered to a subjectafter receiving a transplant. In other embodiments, the compositions areadministered to a subject before and/or simultaneously with and/or afterreceiving a transplant. The compositions of the invention can beadministered to a subject receiving a transplant before the subject hasdeveloped any signs or symptoms of developing GVHD. In this embodiment,the composition modulates the microbiome of the subject in a manner thatprevents or reduces the likelihood that the subject will develop GVHD.In addition or alternatively, the compositions of the invention can beadministered to a subject after the subject has developed GVHD, e.g.,acute GVHD, or chronic GVHD. In this embodiment, the compositionmodulates the microbiome of the subject in a manner that treats orreduces the severity of GVHD. In another embodiment, the composition canbe administered after GVHD has been resolved in order to prevent relapseor recurrence of GVHD.

The microbial compositions, with or without one or more prebiotics,offer a protective and/or therapeutic effect against infection by one ormore GI pathogens of interest and can be administered after an acutecase of infection has been resolved in order to prevent relapse, duringan acute case of infection as a complement to antibiotic therapy if thebacterial composition is not sensitive to the same antibiotics as the GIpathogen, or to prevent infection or reduce transmission from diseasecarriers. In one embodiment, the subject is a transplant recipient. Inanother embodiment, the subject has or is at risk for developing GVHD.

The present microbial compositions, with or without one or moreprebiotics, can be useful in a variety of clinical situations. Forexample, the compositions can be administered as a complementarytreatment to antibiotics when a patient is suffering from an acuteinfection, to reduce the risk of recurrence after an acute infection hassubsided, or when a patient will be in close proximity to others with orat risk of serious gastrointestinal infections (physicians, nurses,hospital workers, family members of those who are ill or hospitalized).

The present microbial compositions, with or without one or moreprebiotics, can be administered to animals, including humans, laboratoryanimals (e.g., primates, rats, mice), livestock (e.g., cows, sheep,goats, pigs, turkeys, chickens), and household pets (e.g., dogs, cats,rodents).

In the present method, the microbial composition, with or without one ormore prebiotics, can be administered enterically, in other words, by aroute of access to the gastrointestinal tract or vagina. This includesoral administration, rectal administration (including enema,suppository, or colonoscopy), by an oral or nasal tube (nasogastric,nasojejunal, oral gastric, or oral jejunal), as detailed more fullyherein.

Pretreatment Protocols.

Prior to administration of the microbial composition, with or withoutone or more prebiotics, the patient can optionally have a pretreatmentprotocol to prepare the gastrointestinal tract or vagina to receive thebacterial composition. In certain embodiments, the pretreatment protocolis advisable, such as when a patient has an acute infection with ahighly resilient pathogen. In other embodiments, the pretreatmentprotocol is entirely optional, such as when the pathogen causing theinfection is not resilient, or the patient has had an acute infectionthat has been successfully treated but where the physician is concernedthat the infection may recur. In these instances, the pretreatmentprotocol can enhance the ability of the bacterial composition to affectthe patient's microbiome.

As one way of preparing the patient for administration of the microbialecosystem, at least one antibiotic can be administered to alter thebacteria in the patient. As another way of preparing the patient foradministration of the microbial ecosystem, a standard colon-cleansingpreparation can be administered to the patient to substantially emptythe contents of the colon, such as used to prepare a patient for acolonoscopy. By “substantially emptying the contents of the colon,” thisapplication means removing at least 75%, at least 80%, at least 90%, atleast 95%, or about 100% of the contents of the ordinary volume of coloncontents. Antibiotic treatment can precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection,or if a patient has received an antibiotic as part of a specificpretreatment protocol, in one embodiment, the antibiotic can be stoppedin sufficient time to allow the antibiotic to be substantially reducedin concentration in the gut or vagina before the bacterial compositionis administered. In one embodiment, the antibiotic can be discontinued1, 2, or 3 days before the administration of the bacterial composition.In another embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or7 antibiotic half-lives before administration of the bacterialcomposition. In another embodiment, the antibiotic can be chosen so theconstituents in the bacterial composition have an MIC50 that is higherthan the concentration of the antibiotic in the gut or vagina.

MIC50 of a bacterial composition or the elements in the composition canbe determined by methods well known in the art. Reller et al.,Antimicrobial Susceptibility Testing: A Review of General Principles andContemporary Practices, Clinical Infectious Diseases 49(11):1749-1755(2009). In such an embodiment, the additional time between antibioticadministration and administration of the bacterial composition is notnecessary. If the pretreatment protocol is part of treatment of an acuteinfection, the antibiotic can be chosen so that the infection issensitive to the antibiotic, but the constituents in the bacterialcomposition are not sensitive to the antibiotic.

Routes of Administration.

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art. Compositionscan be administered by any route suitable for the delivery of disclosedcompositions for treating, inhibiting, or preventing a dysbiosis, ordiseases and disorders associated with a dysbiosis, including, but arenot limited to orally, sublingually, rectally, parentally (e.g.intravenous injection (i.v.), intracranial injection (i.e.);intramuscular injection (i.m.), intraperitoneal injection (i.p.), andsubcutaneous injection (s.c.) and intraosseous infusion (i.o.)),transdermally (using any standard patch), extracorporeally, inhalation,topically or the like, including topical intranasal administration oradministration by inhalant. The compositions and dosage forms describedherein can be administered by e.g., intradermal, ophthalmic,(intra)nasally, local, non-oral, such as aerosol, inhalation,subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal,intra-arterial, and intrathecal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), intravesical, intrapulmonary, intraduodenal,intragastrical, intrabronchial, etc. In preferred embodiments, thepharmaceutical compositions and dosage forms described herein areadministered by routes selected from oral, topical, (trans)dermal,(intra)nasal, and rectal. In certain embodiments, the (intra)nasaladministration is achieved via aerosol or inhalation.

The compositions of the invention are suitable for administration tomammals and non-mammalian animals in need thereof. In certainembodiments, the mammalian subject is a human subject who has one ormore symptoms of a dysbiosis.

In some embodiments, the subject is fed a meal within one hour ofadministration of the probiotic composition. In another embodiment, thesubject is fed a meal concurrently with administration of the probioticcomposition.

When a mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms.

In some embodiments, the gastrointestinal disease, disorder or conditionis a pathogen infection, ulcerative colitis, colitis, Crohn's disease,or irritable bowel disease. Beneficially, the therapeutic composition isadministered only once prior to improvement of the disease, disorder orcondition. In some embodiments, the therapeutic composition isadministered at intervals greater than two days, such as once everythree, four, five or six days, or every week or less frequently thanevery week. In other embodiments, the preparation can be administeredintermittently according to a set schedule, e.g., once a day, onceweekly, or once monthly, or when the subject relapses from the primaryillness. In another embodiment, the preparation may be administered on along-term basis to subjects who are at risk for infection with or whomay be carriers of these pathogens, including subjects who will have aninvasive medical procedure (such as surgery), who will be hospitalized,who live in a long-term care or rehabilitation facility, who are exposedto pathogens by virtue of their profession (livestock and animalprocessing workers), or who could be carriers of pathogens (includinghospital workers such as physicians, nurses, and other health careprofessionals).

In certain embodiments, the microbial composition is administeredenterically. This preferentially includes oral administration, or by anoral or nasal tube (including nasogastric, nasojejunal, oral gastric, ororal jejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy). Themicrobial composition can be administered to at least one region of thegastrointestinal tract, including the mouth, esophagus, stomach, smallintestine, large intestine, and rectum. In some embodiments, it isadministered to all regions of the gastrointestinal tract. The microbialcompositions can be administered orally in the form of medicaments suchas powders, capsules, tablets, gels or liquids. The bacterialcompositions can also be administered in gel or liquid form by the oralroute or through a nasogastric tube, or by the rectal route in a gel orliquid form, by enema or instillation through a colonoscope or by asuppository. In certain embodiments of the above invention, themicrobial composition is administered enterically with one or moreprebiotics.

If the composition is administered colonoscopically and, optionally, ifthe composition is administered by other rectal routes (such as an enemaor suppository) or even if the subject has an oral administration, thesubject can have a colon-cleansing preparation. The colon-cleansingpreparation can facilitate proper use of the colonoscope or otheradministration devices, but even when it does not serve a mechanicalpurpose, it can also maximize the proportion of the bacterialcomposition relative to the other organisms previously residing in thegastrointestinal tract of the subject. For example, the colon cleansingpreparation may maximize the amount of bacterial entities of thebacterial composition that reach and/or engraft in the gastrointestinaltract of the subject. Any ordinarily acceptable colon-cleansingpreparation may be used such as those typically provided when a subjectundergoes a colonoscopy.

Dosages and Schedule for Administration.

The dose administered to a subject should be sufficient to prevent adysbiosis, partially reverse a dysbiosis, fully reverse a dysbiosis, orestablish a healthy-state microbiome. In some aspects, the doseadministered to a subject should be sufficient to prevent the onset ofsymptoms associated with an autoimmune, inflammatory, or barrierdisorder, to reduces the symptoms associated with an autoimmune,inflammatory, or barrier disorder, to eliminate the symptoms associatedwith an autoimmune, inflammatory, or barrier disorder, or to preventrelapse or recurrence of an autoimmune, inflammatory, or barrierdisorder.

One skilled in the art will recognize that dosage will depend upon avariety of factors including the strength of the particular activecomponents employed, as well as the age, species, condition, and bodyweight of the subject. The size of the dose will also be determined bythe route, timing, and frequency of administration as well as theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular composition and the desiredphysiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the active components. Thereafter, the dosageis increased by small increments until the optimum effect under thecircumstances is reached. An effective dosage and treatment protocol canbe determined by routine and conventional means, starting e.g. with alow dose in laboratory animals and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage regimen aswell. Animal studies are commonly used to determine the maximaltolerable dose (“MTD”) of bioactive agent per kilogram weight. Thoseskilled in the art regularly extrapolate doses for efficacy, whileavoiding toxicity, in other species, including humans.

Dosing may be in one or a combination of two or more administrations,e.g., daily, bi-daily, weekly, monthly, or otherwise in accordance withthe judgment of the clinician or practitioner, taking into accountfactors such as age, weight, severity of the disease, and the doseadministered in each administration.

In accordance with the above, in therapeutic applications, the dosagesof the composition used in accordance with the invention vary dependingon the form, depending on the age, weight, and clinical condition of therecipient patient, and depending on the experience and judgment of theclinician or practitioner administering the therapy, among other factorsaffecting the selected dosage. Generally, the dose should be sufficientto result in relieving, and preferably eliminating, a dysbiosis ordisease-associated microbiome, most preferably causing complete recoveryfrom the autoimmune, inflammatory, or barrier disorder. Relief orelimination of a dysbiosis or disease-associated microbiome may bemeasured by culturing and/or sequencing techniques, and well as bydetection of microbial biomarkers in bodily fluids including but notlimited to serum, urine, and feces, or by other techniques known in theart. Relief or elimination of an autoimmune, inflammatory, or barrierdisease, condition, or disorder may be indicated by biopsy andsubsequent analysis of immune cells, microbial cells, and/or TEER, bylocal or systemic measurement of cytokine levels, by detection ofbiomarkers for immune cells, by a lactulose/mannitol test, or by othertechniques known in the art.

In some embodiments, the microbes, carbohydrates, and microbial andprebiotic compositions are provided in a dosage form. In certainembodiments, the dosage form is designed for administration of at leastone OTU or combination thereof disclosed herein, wherein the totalamount of bacterial composition administered is selected from 0. ing to10 g, 10 ng to 1 g, 100 ng to 0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg,or from 10-15 mg. In other embodiments, the bacterial composition isconsumed at a rate of from 0. ing to 10 g a day, 10 ng to 1 g a day, 100ng to 0.1 g a day, 0.1 mg to 500 mg a day, 1 mg to 100 mg a day, or from10-15 mg a day, or more.

In certain embodiments, the treatment period is at least 1 day, at least2 days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, or at least 1 year. Insome embodiments the treatment period is from 1 day to 1 week, from 1week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months,from 6 months to 1 year, or for over a year.

In one embodiment, between about 10⁵ and about 10¹² microorganisms(e.g., CFUs) total can be administered to the patient in a given dosageform. In another embodiment, an effective amount can be provided in from1 to 500 ml or from 1 to 500 grams of the bacterial composition havingfrom 10⁷ to 10¹¹ bacteria per ml or per gram, or a capsule, tablet orsuppository having from 1 mg to 1000 mg lyophilized powder having from10⁷ to 10¹¹ bacteria. Those receiving acute treatment can receive higherdoses than those who are receiving chronic administration (such ashospital workers or those admitted into long-term care facilities).

Any of the preparations described herein can be administered once on asingle occasion or on multiple occasions, such as once a day for severaldays or more than once a day on the day of administration (includingtwice daily, three times daily, or up to five times daily). In anotherembodiment, the preparation can be administered intermittently accordingto a set schedule, e.g., once weekly, once monthly, or when the patientrelapses from the primary illness. In one embodiment, the preparationcan be administered on a long-term basis to individuals who are at riskfor infection with or who may be carriers of these pathogens, includingindividuals who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other health care professionals).

Patient Selection.

Particular microbial compositions, with or without one or moreprebiotic, can be selected for individual patients or for patients withparticular profiles. For example, 16S sequencing can be performed for agiven patient to identify the bacteria present in his or her microbiota.The sequencing can either profile the patient's entire microbiome using16S sequencing (to the family, genera, or species level), a portion ofthe patient's microbiome using 16S sequencing, or it can be used todetect the presence or absence of specific candidate bacteria that arebiomarkers for health or a particular disease state, such as markers ofmulti-drug resistant organisms or specific genera of concern such asEscherichia. Based on the biomarker data, a particular composition canbe selected for administration to a patient to supplement or complementa patient's microbiota in order to restore health or treat or preventdisease. In another embodiment, patients can be screened to determinethe composition of their microbiota to determine the likelihood ofsuccessful treatment.

In some embodiments, metabolite profiles of patient tissue samples ormicrobes cultures from patient tissue are used to identify risk factorsfor developing a gastrointestinal, autoimmune or inflammatory response,to diagnose a gastrointestinal, autoimmune or inflammatory disease, toevaluate the prognosis or severity of said disease, to evaluate thesuccess of a treatment regimen, or any combination thereof. Exemplarymetabolites for the purposes of diagnosis, prognostic risk assessment,or treatment assessment purposes include short chain fatty acids, bileacids, and lactate. In preferred embodiments, metabolite profiles aretaken at different time points during a patient's disease and treatmentin order to better evaluate the patient's disease state includingrecovery or relapse events. Such monitoring is also important to lowerthe risk of a patient developing a new autoimmune condition followingimmunomodulatory treatment. In some embodiments, metabolite profilesinform subsequent treatment, including but not limited to alterations indosage of therapeutic compositions, formations of prebiotic, or theadministration of a particular prebiotic or bacterial population, inorder to promote the growth, proliferation, colonization, and/orengraftment of a desired microbial population in the host. In someembodiments, a patient has a deficiency of a desired microbialpopulation which is enhanced by treatment. In some embodiments, apatient has a excess of a desired microbial population which isdecreased by treatment.

Pharmaceutical Compositions and Formulations of the Invention

Formulations. Provided are formulations for administration to humans andother subjects in need thereof. Generally the microbial compositions arecombined with additional active and/or inactive materials in order toproduce a final product, which may be in single dosage unit or in amulti-dose format. In some embodiments of the invention, the microbialcompositions are comprised of microbes. In some embodiments of theinvention, the microbial compositions are comprised of microbes and oneor more prebiotics.

As described herein, the composition comprises at least one prebioticcarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate can be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates can contain modified saccharideunits, such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates can exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments, the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In other embodiments, thecomposition comprises at least one modified lipid, for example, a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In certain embodiments, the composition comprises at least onesupplemental vitamin. The at least one vitamin can be fat-soluble orwater soluble vitamins. Suitable vitamins include but are not limited tovitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin,niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine,pantothenic acid, and biotin. Suitable forms of any of the foregoing aresalts of the vitamin, derivatives of the vitamin, compounds having thesame or similar activity of the vitamin, and metabolites of the vitamin.

The composition(s) may include different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it needs to be sterile for such routes of administration such asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intlrapericardially, intraumbilically, intraocularally,orally, topically, locally, as an injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in lipid compositions (e.g., liposomes), as anaerosol, or by other method or any combination of the foregoing as wouldbe known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

In other embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In another embodiment, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In cases where a probiotic formulation contains anerobic bacterialstrains, the pharmaceutical formulation and excipients can be selectedto prevent exposure of the bacterial strains to oxygen.

In other embodiments, the composition comprises a binder as anexcipient. Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In another embodiment, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In other embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In other embodiments, the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In another embodiment, the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In another embodiment, the excipient comprises a flavoring agent.Flavoring agents can be chosen from synthetic flavor oils and flavoringaromatics; natural oils; extracts from plants, leaves, flowers, andfruits; and combinations thereof. In some embodiments the flavoringagent is selected from cinnamon oils; oil of wintergreen; peppermintoils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil; and fruitessences including apple, peach, pear, strawberry, raspberry, cherry,plum, pineapple, and apricot.

In other embodiments, the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In yet other embodiments, the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in theformulation is usually about 99% or less, such as about 95% or less,about 90% or less, about 85% or less, about 80% or less, about 75% orless, about 70% or less, about 65% or less, about 60% or less, about 55%or less, 50% or less, about 45% or less, about 40% or less, about 35% orless, about 30% or less, about 25% or less, about 20% or less, about 15%or less, about 10% or less, about 5% or less, about 2% or less, or about1% or less of the total weight of the composition.

The compositions disclosed herein can be formulated into a variety offorms and administered by a number of different means. The compositionscan be administered orally, rectally, or parenterally, in formulationscontaining conventionally acceptable carriers, adjuvants, and vehiclesas desired. The term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, or intrasternal injection and infusiontechniques. In an exemplary embodiment, the composition is administeredorally.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a bacterial compositionand a shell wall that encapsulates the core material. In someembodiments, the core material comprises at least one of a solid, aliquid, and an emulsion. In other embodiments, the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.Suitable polymers include, but are not limited to: cellulosic polymerssuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulosesuccinate and carboxymethylcellulose sodium; acrylic acid polymers andcopolymers, such as those formed from acrylic acid, methacrylic acid,methyl acrylate, ammonio methylacrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate (e.g., those copolymers soldunder the trade name “Eudragit”); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymers; and shellac (purified lac). In yet other embodiments, atleast one polymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed,multiply layered, and/or coated. The coating can be single or multiple.In one embodiment, the coating material comprises at least one of asaccharide, a polysaccharide, and glycoproteins extracted from at leastone of a plant, a fungus, and a microbe. Non-limiting examples includecorn starch, wheat starch, potato starch, tapioca starch, cellulose,hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin,mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gumkaraya, gum ghatti, tragacanth gum, funori, carrageenans, agar,alginates, chitosans, or gellan gum. In some embodiments the coatingmaterial comprises a protein. In another embodiment, the coatingmaterial comprises at least one of a fat and an oil. In otherembodiments, the at least one of a fat and an oil is high temperaturemelting. In yet another embodiment, the at least one of a fat and an oilis hydrogenated or partially hydrogenated. In one embodiment, the atleast one of a fat and an oil is derived from a plant. In otherembodiments, the at least one of a fat and an oil comprises at least oneof glycerides, free fatty acids, and fatty acid esters. In someembodiments, the coating material comprises at least one edible wax. Theedible wax can be derived from animals, insects, or plants. Non-limitingexamples include beeswax, lanolin, bayberry wax, carnauba wax, and ricebran wax. Tablets and pills can additionally be prepared with entericcoatings.

Alternatively, powders or granules embodying the bacterial compositionsdisclosed herein can be incorporated into a food product. In someembodiments, the food product is a drink for oral administration.Non-limiting examples of a suitable drink include fruit juice, a fruitdrink, an artificially flavored drink, an artificially sweetened drink,a carbonated beverage, a sports drink, a liquid diary product, a shake,an alcoholic beverage, a caffeinated beverage, infant formula and soforth. Other suitable means for oral administration include aqueous andnonaqueous solutions, emulsions, suspensions and solutions and/orsuspensions reconstituted from non-effervescent granules, containing atleast one of suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, coloring agents, and flavoringagents.

In some embodiments, the food product can be a solid foodstuff. Suitableexamples of a solid foodstuff include without limitation a food bar, asnack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, afrozen yogurt bar, and the like.

In other embodiments, the compositions disclosed herein are incorporatedinto a therapeutic food. In some embodiments, the therapeutic food is aready-to-use food that optionally contains some or all essentialmacronutrients and micronutrients. In another embodiment, thecompositions disclosed herein are incorporated into a supplementary foodthat is designed to be blended into an existing meal. In one embodiment,the supplemental food contains some or all essential macronutrients andmicronutrients. In another embodiment, the bacterial compositionsdisclosed herein are blended with or added to an existing food tofortify the food's protein nutrition. Examples include food staples(grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea,soda, beer, liquor, sports drinks), snacks, sweets and other foods.

In one embodiment, the formulations are filled into gelatin capsules fororal administration. An example of an appropriate capsule is a 250 mggelatin capsule containing from 10 (up to 100 mg) of lyophilized powder(10⁸ to 10¹¹ bacteria), 160 mg microcrystalline cellulose, 77.5 mggelatin, and 2.5 mg magnesium stearate. In an alternative embodiment,from 10⁵ to 10¹² bacteria may be used, 10⁵ to 10⁷, 10⁶ to 10⁷, or 10⁸ to10¹⁰, with attendant adjustments of the excipients if necessary. In analternative embodiment, an enteric-coated capsule or tablet or with abuffering or protective composition can be used.

The microbial compositions, with or without one or more prebiotics, aregenerally formulated for oral or gastric administration, typically to amammalian subject. In particular embodiments, the composition isformulated for oral administration as a solid, semi-solid, gel, orliquid form, such as in the form of a pill, tablet, capsule, or lozenge.In some embodiments, such formulations contain or are coated by anenteric coating to protect the bacteria through the stomach and smallintestine, although spores are generally resistant to the stomach andsmall intestines. In other embodiments, the microbial compositions, withor without one or more prebiotics, may be formulated with a germinant toenhance engraftment, or efficacy. In yet other embodiments, thebacterial compositions may be co-formulated or co-administered withprebiotic substances, to enhance engraftment or efficacy. In someembodiments, bacterial compositions may be co-formulated orco-administered with prebiotic substances, to enhance engraftment orefficacy.

The microbial compositions, with or without one or more prebiotics, maybe formulated to be effective in a given mammalian subject in a singleadministration or over multiple administrations. For example, a singleadministration is substantially effective to reduce inflammatory andimmune response in a mammalian subject to whom the composition isadministered. Substantially effective means that inflammatory and/orimmune response in the subject is reduced by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99%following administration of the composition. For example, a singleadministration is substantially effective to reduce Cl. difficile and/orCl. difficile toxin content in a mammalian subject to whom thecomposition is administered. Substantially effective means that Cl.difficile and/or Cl. difficile toxin content in the subject is reducedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%or greater than 99% following administration of the composition. In someembodiments, microbial and prebiotic compositions may be formulated asdescribed above.

The composition is formulated such that a single oral dose contains atleast about 1×10⁴ colony forming units of the bacterial entities and/orfungal entities, and a single oral dose will typically contain about1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹²,1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ CFUs of the bacterialentities and/or fungal entities. The presence and/or concentration of agiven type of bacterial may be known or unknown in a given purifiedspore population. If known, for example the concentration of spores of agiven strain, or the aggregate of all strains, is e.g., 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴,1×10¹⁵, or greater than 1×10¹⁵ viable bacterial entities (e.g., CFUs)and/or fungal entities per gram of composition or per administered dose.

In some formulations, the composition contains at least about 0.5%, 1%,2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90%spores on a mass basis. In some formulations, the administered dose doesnot exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacteria and/or fungi may contain a purified population thatincludes a substantial enrichment of bacterial entities present in thefecal material, and wherein the composition optionally comprises agerminant, such as BHIS oxgall, CaDPA, one or more amino acids, a sugar,a nucleoside, a bile salt, a metal or a metal cation, a fatty acid, anda long-chain alkyl amine, or a combination thereof.

It has recently come to light that the DNA of commensal microbes,including many species of Lactobacillus protect against activation oflamina propia dendritic cells and sustain regulatory T cell conversion(Bouladoux N, Hall J A, Grainger J R, dos Santos L M, Kann M G,Nagarajan V, Verthelyi D, and Belkaid Y, 2012. Regulatory role ofsuppressive motifs from commensal DNA. Mucosal Immunol. 5: 623-634).Thus commensal DNA may protect against colitis, IBD, and/or otherimmunological intolerances in the gut. Furthermore, Lactobacillusspecies are prevalent in the healthy vaginal microbiome. Thus, DNA fromLactobacillus or other vaginal microbiome commensals may suppress immuneresponses in the vagina that could disrupt the normal healthy-statevaginal microbiome and lead to complications such as chronic HPV,infertility, miscarriages, or UTIs. As such, in certain embodiments, themicrobial composition, pharmaceutical composition, dosage form, or kitadditionally comprises DNA isolated from one or more host commensals.

Combination Therapy.

The microbial compositions, with or without one or more prebiotics, canbe administered with other agents in a combination therapy mode,including anti-microbial agents. Administration can be sequential, overa period of hours or days, or simultaneous.

In one embodiment, the microbial compositions, with or without one ormore prebiotics, are included in combination therapy with one or moreanti-microbial agents, which include anti-bacterial agents, anti-fungalagents, anti-viral agents and anti-parasitic agents.

Anti-bacterial agents can include cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir,Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol,Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine,Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine,Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine,Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,Zalcitabine, Zanamivir and Zidovudine.

Examples of antifungal compounds include, but are not limited to polyeneantifungals such as natamycin, rimocidin, filipin, nystatin,amphotericin B, candicin, and hamycin; imidazole antifungals such asmiconazole, ketoconazole, clotrimazole, econazole, omoconazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, and tioconazole; triazole antifungals suchas fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, terconazole, and albaconazole; thiazole antifungals suchas abafungin; allylamine antifungals such as terbinafine, naftifine, andbutenafine; and echinocandin antifungals such as anidulafungin,caspofungin, and micafungin. Other compounds that have antifungalproperties include, but are not limited to polygodial, benzoic acid,ciclopirox, tolnaftate, undecylenic acid, flucytosine or5-fluorocytosine, griseofulvin, and haloprogin.

In one embodiment, the bacterial compositions are included incombination therapy with one or more corticosteroids, mesalazine,mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressivedrugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone,methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, and combinations thereof.

In one embodiment, the bacterial compositions are included in acombination or adjuvant therapy with one or more additional treatmentsfor GVHD. For example, the bacterial compositions can be administered toa transplant subject who has been or currently is being treated with animmunosuppressive treatment like cyclosporine, high dose steroids,methotrexate, or methylprednisolone.

A prebiotic is an ingredient that can allow specific changes in both thecomposition and/or activity in the gastrointestinal microbiota thatconfers benefits upon host well-being and health. Prebiotics can includecomplex carbohydrates, amino acids, peptides, or other essentialnutritional components for the survival of the bacterial composition.Prebiotics include, but are not limited to, amino acids, biotin,fructooligosaccharide, galactooligosaccharides, inulin, lactulose,mannan oligosaccharides, oligofructose-enriched inulin, oligofructose,oligodextrose, tagatose, trans-galactooligosaccharide, andxylooligosaccharides.

Methods for Testing Compositions for Populating Effect

In Vivo Assay for Determining Whether a Composition Populates aSubject's Gastrointestinal Tract or Vagina.

In order to determine that the composition populates thegastrointestinal tract or vagina of a subject, an animal model, such asa mouse model, can be used. The model can begin by evaluating themicrobiota of the mice. Qualitative assessments can be accomplishedusing 16S profiling of the microbial community in the feces of normalmice. It can also be accomplished by full genome sequencing, wholegenome shotgun sequencing (WGS), or traditional microbiologicaltechniques. Quantitative assessments can be conducted using quantitativePCR (qPCR), described below, or by using traditional microbiologicaltechniques and counting colony formation.

Optionally, the mice can receive an antibiotic treatment to mimic thecondition of dysbiosis. Antibiotic treatment can decrease the taxonomicrichness, diversity, and evenness of the community, including areduction of abundance of a significant number of bacterial taxa.Dethlefsen et al., The pervasive effects of an antibiotic on the humangut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology6(11):3280 (2008). At least one antibiotic can be used, and antibioticsare well known. Antibiotics can include aminoglycoside antibiotic(amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin, andapramycin), amoxicillin, ampicillin, Augmentin (anamoxicillin/clavulanate potassium combination), cephalosporin (cefaclor,defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime,cefuroxime, cephalexin), clavulanate potassium, clindamycin, colistin,gentamycin, kanamycin, metronidazole, or vancomycin. As an individual,nonlimiting specific example, the mice can be provided with drinkingwater containing a mixture of the antibiotics kanamycin, colistin,gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5mg/kg, 21.5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight),respectively, for 7 days. Alternatively, mice can be administeredciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse bodyweight), for 7 days.

If the mice are provided with an antibiotic, a wash out period of fromone day to three days may be provided with no antibiotic treatment andno bacterial composition treatment.

Subsequently, the composition is administered to the mice by oralgavage. The composition may be administered in a volume of 0.2 mlcontaining 10⁴ CFUs of each type of bacteria in the therapeuticcomposition. Dose-response may be assessed by using a range of doses,including, but not limited to 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,and/or 10¹⁰

The mice can be evaluated using 16S sequencing, full genome sequencing,whole genome shotgun sequencing (WGS), or traditional microbiologicaltechniques to determine whether administering the composition hasresulted in the population by one or more administered bacteria in thegastrointestinal tract or vagina of the mice. For example only, one day,three days, one week, two weeks, and one month after administration ofthe bacterial composition to the mice, 16S profiling is conducted todetermine whether administering the composition has resulted inpopulation by one or more administered bacteria in the gastrointestinaltract or vagina of the mice. Quantitative assessments, including qPCRand traditional microbiological techniques such as colony counting, canadditionally or alternatively be performed, at the same time intervals.

Furthermore, the number of sequence counts that correspond exactly tothose in the composition over time can be assessed to determinespecifically which components of the bacterial composition reside in thegastrointestinal tract or vagina over a particular period of time. Inone embodiment, the bacterial strains of the composition persist for adesired period of time. In another embodiment, the bacterial strains ofthe composition persist for a desired period of time, while alsoincreasing the ability of other microbes (such as those present in theenvironment, food, etc.) to populate the gastrointestinal tract orvagina, further increasing overall diversity, as discussed below.

Ability of Compositions to Populate Different Regions of theGastrointestinal Tract or Vagina.

The present microbial compositions can also be assessed for theirability to populate different regions on the gastrointestinal tract orvagina. In one embodiment, a microbes of the therapeutic composition canbe chosen for its ability to populate one or more than one region of thegastrointestinal tract, including, but not limited to the stomach, thesmall intestine (duodenum, jejunum, and ileum), the large intestine (thececum, the colon (the ascending, transverse, descending, and sigmoidcolon), and the rectum). In another embodiment, the bacterialcomposition can be chosen for its ability to populate one or more thanone region of the vagina. In some embodiments of the above invention,the microbial compositions comprise microbes and one or more prebiotics.

An in vivo study can be conducted to determine which regions of thegastrointestinal tract or vagina a given bacterial composition willpopulate. A mouse model similar to the one described above can beconducted, except instead of assessing the feces produced by the mice,particular regions of the gastrointestinal tract or vagina can beremoved and studied individually. For example, at least one particularregion of the gastrointestinal tract or vagina can be removed and aqualitative or quantitative determination can be performed on thecontents of that region of the gastrointestinal tract or vagina. Inanother embodiment, the contents can optionally be removed and thequalitative or quantitative determination may be conducted on the tissueremoved from the mouse.

qPCR.

As one quantitative method for determining whether a microbialcomposition, with or without one or more prebiotics, populates thegastrointestinal tract or vagina, quantitative PCR (qPCR) can beperformed. Standard techniques can be followed to generate a standardcurve for the bacterial composition of interest, either for all of thecomponents of the bacterial composition collectively, individually, orin subsets (if applicable). Genomic DNA can be extracted from samplesusing commercially-available kits, such as the Mo Bio Powersoil®-htp 96Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), theMo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad,Calif.), or the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.)according to the manufacturer's instructions.

In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME,Gaithersburg, Md.) and primers specific for the bacterial composition ofinterest, and may be conducted on a MicroAmp® Fast Optical 96-wellReaction Plate with Barcode (0. mL) (Life Technologies, Grand Island,N.Y.) and performed on a BioRad C1000™ Thermal Cycler equipped with aCFX96™ Real-Time System (BioRad, Hercules, Calif.), with fluorescentreadings of the FAM and ROX channels. The Cq value for each well on theFAM channel is determined by the CFX Manager™ software version 2.1. Thelog₁₀(cfu/ml) of each experimental sample is calculated by inputting agiven sample's Cq value into linear regression model generated from thestandard curve comparing the Cq values of the standard curve wells tothe known log₁₀(cfu/ml) of those samples. The skilled artisan may employalternative qPCR modes.

VIII. Distal Dysbiosis

The probiotic compositions described herein have beneficial effects forthe subject locally, at the site of administration (e.g., in thegastrointestinal tract for compositions administered orally, or in thevagina for compositions administered vaginally), as previouslydescribed. Surprisingly, the probiotic compositions described herein mayalso be used to correct or prevent a dysbiosis at a site distal to thesite of administration.

“Dysbiosis” refers to a state of the microbiota of the gut or other bodyarea in a subject, including mucosal or skin surfaces in which thenormal diversity and/or function of the ecological network is disrupted.This unhealthy state can be due to a decrease in diversity, theovergrowth of one or more pathogens or pathobionts, symbiotic organismsable to cause disease only when certain genetic and/or environmentalconditions are present in a subject, or the shift to an ecologicalmicrobial network that no longer provides an essential function to thehost subject, and therefore no longer promotes health. Accordingly, a“gastrointestinal dysbiosis” refers to a state of the microbiota ormicrobiome of the gut in which the normal diversity and/or function ofthe ecological network or niche is disrupted. The term “gut” as usedherein is meant to refer to the entire gastrointestinal or digestivetract (also referred to as the alimentary canal) and it refers to thesystem of organs within multi-cellular animals which takes in food,digests it to extract energy and nutrients, and expels the remainingwaste. As used herein the term “gastrointestinal tract” refers to theentire digestive canal, from the oral cavity to the rectum. The term“gastrointestinal tract” includes, but is not limited to, mouth andproceeds to the esophagus, stomach, small intestine, large intestine,rectum and, finally, the anus.

The term “distal” generally is used in relation to the gastrointestinaltract, specifically the intestinal lumen, of a human or other mammal,which represent the intended sites of engraftment or colonization forprobiotics administered orally. Thus, in relation to probioticsadministered to the gastrointestinal tract, a “distal dysbiosis”includes a dysbiosis outside of the lumen of the gastrointestinal tract.In other instances, the term “distal” may be used in relation to thesite of administration, intended engraftment, or intended colonizationof a composition, e.g., a probiotic composition, of the invention. Forexample, if a probiotic composition is administered vaginally, a distaleffect of the composition would occur outside the vagina. Similarly, ifa probiotic composition is administered to the skin, e.g., through askin patch, transdermal lotion, etc., a distal effect of the compositionwould occur in a niche other than the skin. If a probiotic compositionis administered to the lungs, e.g., in an inhalable formulation, adistal effect of the composition would occur outside the lungs. If aprobiotic composition is administered to the ear, eye, nose, etc., adistal effect of the composition would occur at a site other than thesite of administration, engraftment, or colonization of the composition(i.e., distal to the ear, distal to the eye, distal to the nose, etc.).

Distal sites include but are not limited to the liver, spleen, fallopiantubes and uterus. Other distal sites include skin, blood and lymphnodes. In other embodiments, the distal site is placenta, spleen, liver,uterus, blood, eyes, ears, lungs, liver, pancreas, brain, embryonic sac,or vagina. In another embodiment, the distal site is vagina, skin,lungs, brain, nose, ear, eyes/conjunctiva, mouth, circulatory system,e.g., blood, placenta, reproductive tract, cardiovascular system, and/ornervous system. A probiotic composition may have an effect on themicrobiota of more than one distal site in a subject. For example, insome embodiments, a probiotic composition modulates the microbiota ofone or more sites distal to the site of administration, engraftment, orcolonization, e.g., one or more of placenta, spleen, liver, uterus,blood, eyes, ears, lungs, liver, pancreas, brain, embryonic sac, vagina,skin, brain, nose, mouth, reproductive tract, cardiovascular system,and/or nervous system.

Any disruption from a preferred (e.g., ideal, normal, or beneficial)state of the microbiota can be considered a dysbiosis, even if suchdysbiosis does not result in a detectable disease or disorder, ordecrease in health. This state of dysbiosis may lead to a disease ordisorder (e.g. GVHD, transplant rejection, and related conditions), orthe state of dysbiosis may lead to a disease or disorder (e.g. GVHD,transplant rejection, and related conditions) only under certainconditions, or the state of dysbiosis may prevent a subject fromresponding to treatment or recovering from a disease or disorder (e.g.GVHD, transplant rejection, and related conditions). In the case ofGVHD, a gastrointestinal dysbiosis can contribute to the pathology ofGVHD by increasing inflammation and/or reducing intestinal barrierintegrity. A dysbiosis distal to the gastrointestinal tract can alsocontribute to GVHD pathology, for example, by increasing systemicinflammation in the subject. In one embodiment, the distal dysbiosis isat or near the site of the transplant. Accordingly, probioticcompositions of the invention that modulate the microbiome, e.g., tocorrect a dysbiosis, can be used to prevent or treat GVHD in atransplant recipient. In addition, probiotic compositions that reduceinflammation and/or increase intestinal barrier integrity can be used toprevent or treat GVHD in a transplant recipient.

In certain aspects, the present invention is directed to a method ofreconstituting, modulating, or creating a beneficial bacterial flora inthe gastrointestinal tract of a mammalian host in need thereof,comprising administering to the mammalian host a composition comprisingat least one isolated bacterial population. In one embodiment, the atleast one bacterial population is coadministered or coformulated withone or more prebiotic, e.g, at least one polymer or monomer. In oneembodiment, the prebiotic is a carbohydrate, e.g., xylose. In oneembodiment, the subject is a transplant recipient. In one embodiment,the subject has or is at risk for developing GVHD. In certainembodiments the gastrointestinal disease, disorder or condition is adisease or disorder associated with or characterized by reducedintestinal integrity.

In certain other aspects, the present invention is directed to a methodof treating or alleviating a transplant disorder, e.g., GVHD ortransplant rejection, in a subject in need thereof, the methodcomprising a administering to the subject at least one isolatedbacterial population. In one embodiment, the at least one bacterialpopulation is coadministered or coformulated with one or more prebiotic,e.g, at least one polymer or monomer. In one embodiment, the one or moreprebiotic is a carbohydrate, e.g., xylose.

Provided are compositions and methods to provide modulation, engraftmentand/or augmentation of one or more bacterial and/or fungal entities to adistal site. In order to characterize the alteration of a target niche,such as by engraftment and/or augmentation of a bacteria within theniche, provided are methods of detecting, quantifying and characterizing16S, 18S and ITS signatures in skin, vagina, etc. Moreover, provided aremethods of detecting bacterial and fungal components typicallyassociated with one microbiota in a distal site, often associating with(in a physiological or manner) with the microbiota of that distal site.For example, following administration of a composition, bacteriadetectably present in the GI tract or vagina prior to administration aredetected in distal sites, for example, the blood, or another nicheoutside the GI lumen. For example, changes in the microbiome at a givensite (e.g. GI tract) lead to changes in the microbiome at a distal site(e.g. vagina).

Accordingly, detecting and quantifying 16S, 18S and ITS signatures ofthe microbial network at a distal site can be used to characterize thecomponents of the microbiome at the distal site under normal, healthyconditions, and can also be used to detect a dysbiosis at the distalsite, when the components of the microbiome at the distal site aredisrupted.

In order to characterize a distal dysbiosis, provided are methods ofdetecting, quantifying and characterizing 16S, 18S and ITS signatures inimmune organs, such as the lymph nodes, spleen, etc. Moreover, providedare methods of detecting bacterial and fungal components typicallyassociated with one microbiota in a distal site, often associating (in aphysiological or pathological manner) with the microbiota of that distalsite. For example, bacteria normally detected in the GI tract or vaginaare detected in distal sites, for example, the blood.

A distal dysbiosis includes disruptions in the normal diversity and/orfunction of the microbial network in a subject at a site other than thegastrointestinal tract, which is generally the site of administration ofprobiotics provided orally. In cases where a probiotic composition isadministered to a site other than the gastrointestinal tract, a distaldysbiosis can include disruptions in the normal diversity and/orfunction of the microbial network in a subject at a site other than thesite of administration, colonization or engraftment.

Probiotic compositions described herein can correct or treat a distaldysbiosis by correcting the imbalance in microbial diversity that ispresent at the distal site. Bacteria contained in the probioticcomposition can correct the distal dysbiosis directly, by translocatingto the distal site. Bacteria contained in the probiotic composition canalso correct the distal dysbiosis indirectly, by promoting translocationof other gut commensals to the distal site, or by modifying themicroenvironment of the distal site to create conditions that restore ahealthy microbiome, e.g., by reducing inflammation.

Without wishing to be bound by theory, the probiotic compositions of theinvention may impact distal sites in several ways.

In one embodiment, a bacterial strain present in the probioticcomposition engrafts in the gastrointestinal tract of a subject, andtranslocates to a distal site, thereby augmenting the bacterial strainpresent in the probiotic composition at the distal site. In oneembodiment, the bacterial strain present in the probiotic composition isnot detectably present at the distal site prior to administration of theprobiotic.

In another embodiment, a bacterial strain present in the probioticcomposition is augmented in the gastrointestinal tract of a subjectwithout engraftment, and translocates to a distal site, therebyaugmenting the bacterial strain present in the probiotic composition atthe distal site. In one embodiment, the bacterial strain present in theprobiotic composition is not detectably present at the distal site priorto administration of the probiotic.

In another embodiment, a bacterial strain present in the probioticcomposition modulates the microenvironment of the gut, augmenting asecond bacterial strain present within the gut microbiota. The secondbacterial strain augmented in the gut translocates to a distal site,thereby augmenting the second bacterial strain at the distal site. Inembodiments, the second bacterial strain is not present in the probioticcomposition. In some embodiments, the bacterial strain present in theprobiotic composition is an immunomodulatory bacteria, e.g., ananti-inflammatory bacteria. Modulation of the microenvironment of thegut may include, for example, alteration of cytokines secreted by hostcells in and around the gut, reducing inflammation in the gut,increasing secretion of short chain fatty acids in the gut, or alteringthe proportion of immune cell subpopulations in the gut, each of whichimpacts the gut microbiome. Modulation of the microenvironment of thegut can include increasing or decreasing overall microbial diversity.

In another embodiment, a bacterial strain present in the probioticcomposition modulates the microenvironment at a distal site in asubject, thereby augmenting a second bacterial strain at the distalsite. In embodiments, the second bacterial strain is not present in theprobiotic composition. In some embodiments, the bacterial strain presentin the probiotic composition is an immunomodulatory bacteria, e.g., ananti-inflammatory bacteria. Immunomodulatory bacteria can modulate themicroenvironment at a site distal to the gastrointestinal tract in asubject by, for example, reducing systemic inflammation. This can beachieved by altering the profile of cytokine expression by immune cells,or altering the proportion of immune cell subpopulations. Bacterialstrains present in the probiotic composition can also modulateintestinal permeability, e.g., by secretion of short chain fatty acids,which impacts the microenvironment of distal sites. In addition oralternatively, bacterial strains present in the probiotic compositioncan increase or decrease overall microbial diversity.

Accordingly, the probiotic compositions described herein may additivelyor synergistically elicit an immunomodulatory response either distally,e.g., in which enteral administration of microbes results in alteringthe immune response at a site outside the gastrointestinal tract such asthe skin or liver, or locally, e.g. the enteral administration ofmicrobes results in altering the immune response in the gastrointestinaltract, e.g., in the intestines.

The immune system of a subject and the microbiome of the subject areclosely linked, and interact systemically. Disruptions to themicrobiome, both in the gastrointestinal tract and at distal sites, canhave profound effects throughout the body of the subject. In particular,disruptions to the microbiome increase systemic inflammation andintestinal barrier dysfunction in a subject. Increased inflammation andintestinal barrier dysfunction negatively impact the health of thesubject in many ways, by contributing to a wide range of inflammatoryand autoimmune conditions distal to the gastrointestinal tract.Conversely, increased inflammation in a subject leads to disruptions inthe subject's microbiome, and disruptions to the microbiome lead in turnto further increases in inflammation. Administration of a probioticcomposition containing immunomodulatory bacteria can reduce inflammationin the gastrointestinal tract and restore intestinal barrier integrity,resulting in a reduction in inflammation at sites distal to thegastrointestinal tract, and improvement in the symptoms of autoimmune orinflammatory disorders associated with systemic inflammation.Administration of a probiotic composition containing bacterial strainsthat secrete short chain fatty acids are also capable of reducinginflammation restoring intestinal barrier integrity.

In other embodiments, the probiotic compositions of the inventionimprove blood/brain barrier integrity. In other embodiments, theprobiotic compositions of the invention improve lung epitheliumintegrity.

The probiotic compositions and methods described herein can prevent ortreat the loss or reduction of barrier function recognized to occurduring dysbiosis or in the shift in one or more microbiotal populationsthat give rise to the dysbiosis. The loss of barrier function results insystemic seeding of bacterial populations resulting in dysbioticactivity, and in some events, the loss of barrier function results in alocal reseeding of the bacterial populations. In both situations, theresulting immune activation leads to pathogenic inflammatory and immuneresponses. In response, provided are compositions that are capable ofrestoring barrier function, restoring the normal microbiotal components,and reducing (e.g., suppressing) immune/inflammatory response. In oneembodiment, the improvement of gut epithelium barrier integrity resultsin reduced trafficking of bacteria, bacterial components and/orbacterial metabolites into the blood. In some compositions, provided areantibiotic agents that remove the existing microflora in a target niche,while newly administered or recruited bacteria and fungi populate (orre-populate) the target niche. The combination with carbohydrates (e.g.,by co-administration or co-formulation) may synergistically affect thispopulation/repopulation technique.

Disorders associated with a dysbiosis, i.e., a gastrointestinaldysbiosis or a distal dysbiosis, which increases systemic inflammationand/or reduces intestinal barrier integrity include, for example,autoimmune or inflammatory disorders, Crohn's Disease, vaginaldysbiosis, and transplant disorders such as graft-versus-host disease.These disorders can be treated by administration (e.g., oraladministration) of probiotic compositions containing immunomodulatory(e.g., anti-inflammatory) bacterial strains.

In some embodiments, the probiotic compositions described herein mayadditively or synergistically reduce the number of types of autoimmunedisease- or inflammatory disease-associated pathogens or pathobiontseither distally—e.g., orally-administered microbes reduce the totalmicrobial burden in an organ not in the gastrointestinal tract, orintravaginally-administered microbes reduce the total microbial burdenin an organ that is not the vagina—or locally, e.g., the intestines orvagina, respectively.

Accordingly, in one aspect, the invention provides a method of reducinginflammation in a subject, comprising administering to the subject aprobiotic composition comprising an isolated, anti-inflammatorybacterial population, such that inflammation in the subject is reduced.A systemic reduction in inflammation can modulate the microbiome ofniches distal to the site of administration, intended engraftment, orintended colonization of the bacterial population. The probioticcomposition can contain an excipient useful for formulation as apharmaceutical composition. In instances where the bacterial populationincludes anaerobic bacteria, the excipient can, in one embodiment,reduce exposure of the bacterial population to oxygen.

In a preferred embodiment, administration of the probiotic compositioncan reduce inflammation at a site distal to the site of administration,engraftment, or colonization, such as, for example, vagina, skin, lungs,brain, nose, ear, eyes/conjunctiva, mouth, circulatory system, e.g.,blood, placenta, embryonic sac, reproductive tract, cardiovascularsystem, and/or nervous system. In one embodiment, administration of theprobiotic composition can reduce inflammation at a site selected fromblood, skin, vagina, liver, spleen, fallopian tubes, uterus, or acombination thereof. In one embodiment, administration of the probioticcomposition modulates the microbiome at a distal site.

The anti-inflammatory bacterial population can induce a decrease insecretion of pro-inflammatory cytokines and/or an increase in secretionof anti-inflammatory cytokines by host cells. The anti-inflammatoryproperties of the bacterial population can be determined by methodsdescribed herein or known in the art, for example, by measuringalterations in cytokine secretion by peripheral blood mononuclear cells(PBMCs) exposed to the bacterial population. Anti-inflammatory bacteriacan be selected for inclusion in the probiotic formulation based onmodulation of particular cytokines of interest. For example,anti-inflammatory bacteria can be selected based on the ability todecrease secretion of one or more pro-inflammatory cytokines, e.g.,IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, andcombinations thereof, and/or the ability to increase secretion of one ormore anti-inflammatory cytokines, e.g., IL-10, IL-13, IL-4, IL-5, andcombinations thereof.

In another aspect, the invention provides methods of treating orpreventing a distal dysbiosis in a subject, by administering to thesubject a probiotic composition comprising an isolated bacterialpopulation in an amount sufficient to alter the microbiome at a sitedistal to the site of administration, engraftment, or colonization ofthe bacterial population, such that the distal dysbiosis is treated. Forexample, administration of the probiotic composition may modulate afirst microbiome at the site of administration, engraftment orcolonization of the bacterial population, causing subsequent modulationof a second microbiome at a site that is distinct from the firstmicrobiome, e.g., a distal site.

In one embodiment, the invention provides methods of treating orpreventing a distal dysbiosis, by orally administering a probioticcomposition which alters the microbiome at a site distal to thegastrointestinal tract.

In another aspect, the invention provides a method of treating orpreventing a disorder associated with a distal dysbiosis in a subject inneed thereof, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population in an amountsufficient to alter the microbiome at a site of the distal dysbiosis,such that the disorder associated with the distal dysbiosis is treated.Disorders associated with distal dysbiosis, including disruptions to thesystemic microbiome, are described herein and include, for example,autoimmune or inflammatory disorders such as graft-versus-host disease(GVHD), an inflammatory bowel disease (IBD), ulterative colitis, Crohn'sdisease, multiple sclerosis (MS), systemic lupus erythematosus (SLE),type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and Celiacdisease; transplant disorders such as graft-versus-host disease; andvaginal dysbiosis. In one embodiment, the disorder associated withdistal dysbiosis occurs in the respiratory tract (e.g., lung), includingbut not limited to Cystic Fibrosis and chronic obstructive pulmonarydisorder (COPD).

In one embodiment, the probiotic composition contains a species ofbacteria that is deficient at the site of the distal dysbiosis.Administration of the probiotic composition can increase the quantity ofthe deficient species in the distal microbiome. In one embodiment, thedeficient species is not detectably present at the site of the distaldysbiosis prior to administration of the probiotic composition. In oneembodiment, the species of bacteria in the probiotic compositiontranslocates to the site of the distal dysbiosis.

In another embodiment, the probiotic composition results in augmentationof a species of bacteria not present in the probiotic composition at adistal site. This augmentation can result from, for example,translocation of a species of bacteria not present in the probioticcomposition to the distal site, and/or modulation of themicroenvironment of the distal site in a manner that alters themicrobiome.

In preferred embodiments, the probiotic composition containsimmunomodulatory bacteria, e.g., anti-inflammatory bacteria.

In another aspect, the invention provides a method of reducingintestinal permeability in a subject, by administering a probioticcomposition comprising an isolated bacterial population, whereinadministration of the probiotic composition augments a species ofbacteria that produces short chain fatty acids, such that the intestinalpermeability of the subject is reduced. In other embodiments, intestinalpermeability and disorders associated therewith is improved byadministering a probiotic composition containing mucin-containingbacteria, and/or anti-inflammatory bacteria.

Probiotic compositions useful for correcting or treating a distaldysbiosis, or for treating a disorder distal to the gastrointestinaltract associated with a dysbiosis, can include any of the probioticcompositions described herein. In exemplary embodiments, a probioticcomposition useful for correcting or treating a distal dysbiosisincludes one or more bacterial strains from Table 1. In otherembodiments, the probiotic composition useful for correcting or treatinga distal dysbiosis includes one or more bacterial strains from Table 1A.In other embodiments, the probiotic composition useful for correcting ortreating a distal dysbiosis includes one or more bacterial strains fromTable 1B. In other embodiments, the probiotic composition useful forcorrecting or treating a distal dysbiosis includes one or more bacterialstrains from Table 1C. In other embodiments, the probiotic compositionuseful for correcting or treating a distal dysbiosis includes one ormore bacterial strains from Table 1D. In other embodiments, theprobiotic composition useful for correcting or treating a distaldysbiosis includes one or more bacterial strains from Table 1E. In otherembodiments, the probiotic composition useful for correcting or treatinga distal dysbiosis includes one or more bacterial strains from Table 1F.In some embodiments, the probiotic composition contains a single strainof bacteria. In other embodiments, the probiotic composition containstwo or more strains of bacteria, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more strains ofbacteria. In other embodiments, the probiotic composition contains or isadministered in conjunction with a prebiotic, as described herein.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter.

Preferred bacterial genera also include Acetonema, Alkaliphilus,Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor,Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella,Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Halobacteroides, Heliobacterium, Heliophilum, Heliorestis,Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.),Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora,Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum,Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus,Thermoanaerobacter, Thermosinus and Heliobacillus.

As provided herein, therapeutic compositions comprise, or in thealternative, modulate, the colonization and/or engraftment, of thefollowing exemplary bacterial entities: Lactobacillus gasseri,Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis,Enterococcus durans, Enterococcus villorum, Lactobacillus plantarum,Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcuscohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcusmitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcusanginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridiumsaccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6,Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcusgnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautiaproducta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonellaparvula, Clostridium methylpentosum, Clostridium islandicum,Faecalibacterium prausnitzii, Bacteroides uniformmis, Bacteroidesthetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus,Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirumpropionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria,Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta.

Preferred bacterial species are provided in Table 1, Table 1A, Table 1B,Table 1C, Table 1D, Table 1E, Table 1F, and Table 5. Optionally, in someembodiments, preferred bacterial species are spore formers. Thebacterial species may be used in vegetative form and/or in spore form.Thus, in some embodiments, the bacteria present in a composition aresolely in spore form. In some embodiments, the bacteria present in acomposition are solely in vegetative form. In some embodiments, thebacteria present in a composition are in a combination of vegetativeform and spore form. Where specific strains of a species are provided,one of skill in the art will recognize that other strains of the speciescan be substituted for the named strain.

In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Acidaminococcusintestine. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isAcinetobacter baumannii. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Acinetobacter lwoffii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Akkermansia muciniphila. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Alistipes putredinis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Alistipes shahii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerostipes hadrus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerotruncus colihominis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides caccae.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidescellulosilyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isBacteroides dorei. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides eggerthii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides finegoldii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides fragilis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroides massiliensis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides ovatus.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidessalanitronis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBacteroides salyersiae. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides sp. 1_1_6. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides sp. 3_1_23. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides sp. D20. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroidesthetaiotaomicrond. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides uniformis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides vulgatus. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bifidobacterium adolescentis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacterium bifidum. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Bifidobacterium breve.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bifidobacteriumfaecale. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBifidobacterium kashiwanohense. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Bifidobacterium longum subsp. Longum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bifidobacterium stercoris. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Blautia (Ruminococcus) coccoides. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia faecis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia glucerasea. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)hansenii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)luti. In one embodiment, the bacterial entity, e.g., species or strain,useful in the compositions and methods of the invention is Blautia(Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia producta (Ruminococcus productus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)schinkii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia stercoris. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1).In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Blautia unculturedbacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia wexlerae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Candidatus Arthromitus sp.SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isCatenibacterium mitsuokai. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridiales bacterium1_7_47FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium asparagiforme. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridium bolteae. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium clostridioforme. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium glycyrrhizinilyticum. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium (Hungatella)hathewayi. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium histolyticum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium indolis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium leptum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium (Tyzzerella) nexile. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium perfringens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium(Erysipelatoclostridium) ramosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium scindens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium septum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium sp. 14774. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium sp.7_3_54FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium sp. HGF2. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium symbiosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Collinsella aerofaciens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Collinsella intestinalis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Coprobacillus sp. D7. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus catus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus comes. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Dorea formicigenerans.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Dorea longicatena.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Enterococcusfaecalis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEnterococcus faecium. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli S88. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacterium eligens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacteriumfissicatena. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEubacterium ramulus. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Eubacterium rectale. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Faecalibacterium prausnitzii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Flavonifractor plautii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Fusobacterium mortiferum.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Fusobacteriumnucleatum. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isHoldemania filiformis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Hydrogenoanaerobacterium saccharovorans. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Klebsiella oxytoca. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isLachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Lachnospiraceae bacterium 5_1_57FAA. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus casei. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus rhamnosus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Lactobacillus ruminis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Lactococcus casei.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Odoribactersplanchnicus. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isOscillibacter valericigenes. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Parabacteroides gordonii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Parabacteroides johnsonii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Parabacteroides merdae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Pediococcus acidilactici.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Peptostreptococcusasaccharolyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isPropionibacterium granulosum. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Roseburia intestinalis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Roseburia inulinivorans. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Ruminococcus faecis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus gnavus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus sp. ID8. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Ruminococcus torques.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Slackia piriformis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Staphylococcusepidermidis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStaphylococcus saprophyticus. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Streptococcus cristatus. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus dysgalactiae subsp. Equisimilis. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Streptococcus infantis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Streptococcusoralis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStreptococcus sanguinis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Streptococcus viridans. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus thermophiles. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Veillonella dispar.

In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Acidaminococcus intestine. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter baumannii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter lwoffii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Akkermansia muciniphila. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes putredinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes shahii. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Anaerostipes hadrus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Anaerotruncus colihominis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Bacteroides caccae. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides cellulosilyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides dorei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides eggerthii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides finegoldii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides fragilis. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Bacteroidesmassiliensis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Bacteroides ovatus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bacteroides salanitronis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides salyersiae. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 1_1_6. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 3_1_23. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. D20. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides thetaiotaomicrond. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides uniformis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides vulgatus. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium adolescentis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium bifidum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium breve. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium faecale. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium kashiwanohense. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium longum subsp. Longum.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Bifidobacteriumstercoris. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) coccoides. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia faecis. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiaglucerasea. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) hansenii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) luti. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) obeum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia producta (Ruminococcusproductus). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) schinkii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia stercoris. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiauncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia uncultured bacterium cloneSJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia wexlerae. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Catenibacterium mitsuokai. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridiaceae bacterium (Dielmafastidiosa) JC13. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Clostridialesbacterium 1_7_47FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumasparagiforme. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium bolteae.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium clostridioforme. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium glycyrrhizinilyticum. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Hungatella) hathewayi.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium histolyticum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium indolis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium leptum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Tyzzerella) nexile. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium perfringens. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Erysipelatoclostridium)ramosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumscindens. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium septum. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumsp. 14774. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.7_3_54FAA. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.HGF2. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumsymbiosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaaerofaciens. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaintestinalis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprobacillus sp.D7. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprococcus catus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Coprococcus comes. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea formicigenerans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea longicatena. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecalis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecium. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Erysipelotrichaceae bacterium 3_1_53. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Escherichia coli. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Escherichia coli S88. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium eligens. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium fissicatena. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium ramulus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium rectale. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Faecalibacterium prausnitzii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Flavonifractor plautii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium mortiferum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium nucleatum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Holdemania filiformis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Hydrogenoanaerobacterium saccharovorans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Klebsiella oxytoca. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Lachnospiraceaebacterium 7_1_58FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprisesLachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus casei. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus rhamnosus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus ruminis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactococcus casei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOdoribacter splanchnicus. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOscillibacter valericigenes. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides gordonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides johnsonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides merdae. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPediococcus acidilactici. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPeptostreptococcus asaccharolyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Propionibacterium granulosum. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia intestinalis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia inulinivorans. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus faecis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus gnavus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus sp. ID8. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus torques. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Slackia piriformis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus epidermidis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus saprophyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus cristatus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus dysgalactiae subsp. Equisimilis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus infantis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus oralis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus sanguinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus viridans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus thermophiles. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Veillonella dispar.

Exemplary probiotic compositions useful for treatment of disordersassociated with a distal dysbiosis contain bacterial strains capable ofreducing inflammation in a subject. As described herein, suchimmunomodulatory (anti-inflammatory) bacteria can modulate cytokineexpression by host immune cells, resulting in an overall increase insecretion of anti-inflammatory cytokines and/or an overall decrease insecretion of pro-inflammatory cytokines, systemically reducinginflammation in the subject. In exemplary embodiments, probioticcompositions useful for treatment of disorders associated with a distaldysbiosis stimulate secretion of one or more anti-inflammatory cytokinesby host immune cells, such as PBMCs. Anti-inflammatory cytokinesinclude, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ,and combinations thereof. In other exemplary embodiments, probioticcompositions useful for treatment of disorders associated with a distaldysbiosis inhibit secretion of one or more pro-inflammatory cytokines byhost immune cells, such as PBMCs. Pro-inflammatory cytokines include,but are not limited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α,MIP1β, TNFα, and combinations thereof. Other exemplary cytokines areknown in the art and are described herein. Probiotic compositionscontaining anti-inflammatory bacteria reduce inflammation at the site ofadministration, e.g., in the gastrointestinal tract, as well as atdistal sites throughout the body of the subject.

Other exemplary probiotic compositions useful for treatment of disordersassociated with a dysbiosis distal to the gastrointestinal tract containbacterial strains capable of altering the proportion of immunesubpopulations, e.g., T cell subpopulations, in the subject.

For example, immunomodulatory bacteria can increase or decrease theproportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in asubject. The increase or decrease in the proportion of immune cellsubpopulations may be systemic, or it may be localized to a site ofaction of the probiotic, e.g., in the gastrointestinal tract or at thesite of a distal dysbiosis. In some embodiments, a probiotic compositioncomprising immunomodulatory bacteria is used for treatment of disordersassociated with a dysbiosis distal to the gastrointestinal tract basedon the desired effect of the probiotic composition on thedifferentiation and/or expansion of subpopulations of immune cells inthe subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria that increase the proportion of Treg cells in a subject. Inanother embodiment, a probiotic composition contains immunomodulatorybacteria that decrease the proportion of Treg cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th17 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th17 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th1 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th1 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th2 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th2 cells in a subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria capable of modulating the proportion of one or more of Tregcells, Th17 cells, Th1 cells, and combinations thereof in a subject.Certain immune cell profiles may be particularly desirable to treat orprevent particular disorders associated with a dysbiosis. For example,treatment or prevention of autoimmune or inflammatory disorders can bepromoted by increasing numbers of Treg cells and Th2 cells, anddecreasing numbers of Th17 cells and Th1 cells. Accordingly, probioticcompositions for the treatment or prevention of autoimmune orinflammatory disorders may contain probiotics capable of promoting Tregcells and Th2 cells, and reducing Th17 and Th1 cells.

Short chain fatty acids (SCFAs) can have immunomodulatory (i.e.,immunosuppressive) effects and therefore their production (i.e.,biosynthesis or conversion by fermentation) is advantageous for theprevention, control, mitigation, and treatment of autoimmune and/orinflammatory disorders (Lara-Villoslada F. et al., 2006. Short-chainfructooligosaccharides, in spite of being fermented in the upper part ofthe large intestine, have anti-inflammatory activity in the TNBS modelof colitis. Eur J Nutr. 45(7): 418-425). In germ-free mice andvancomycin-treated conventional mice, administration of SCFA (acetate,propionate, or butyrate) restored normal numbers of Tregs in the largeintestine (Smith P M, et al. Science. 2013; 569-573). Short-chain fattyacids (SCFA) are produced by some bacteria as a byproduct of xylosefermentation. SCFA are one of the most abundant metabolites produced bythe gut microbiome, particularly the family Clostridiacea, includingmembers of the genus Clostridium, Ruminococcus, or Blautia. In someaspects, the pharmaceutical composition, dosage form, or kit comprisesat least one type of microbe (e.g., one or more microbial species, suchas a bacterial species, or more than one strain of a particularmicrobial species) and at least one type of prebiotic such that thecomposition, dosage form, or kit is capable of increasing the level ofone or more immunomodulatory SCFA (e.g., acetate, propionate, butyrate,or valerate) in a mammalian subject. Optionally, the pharmaceuticalcomposition, dosage form, or kit further comprises one or moresubstrates of one or more SCFA-producing fermentation and/orbiosynthesis pathways. In certain embodiments, the administration of thecomposition, dosage form, or kit to a mammalian subject results in theincrease of one or more SCFAs in the mammalian subject by approximately1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, orgreater than 100-fold. In some embodiments, the dysbiosis is caused by adeficiency in microbes that produce short chain fatty acids.Accordingly, in some embodiments, the probiotic composition can containa species of bacteria that produce short chain fatty acids.

Aspects of this invention also include medium chain triglycerides(MCTs). MCTs passively diffuse from the GI tract to the portal system(longer fatty acids are absorbed into the lymphatic system) withoutrequirement for modification like long-chain fatty acids orvery-long-chain fatty acids. In addition, MCTs do not require bile saltsfor digestion. Patients who have malnutrition or malabsorption syndromesare treated with MCTs because they do not require energy for absorption,use, or storage. Medium-chain triglycerides are generally considered agood biologically inert source of energy that the human body findsreasonably easy to metabolize. They have potentially beneficialattributes in protein metabolism, but may be contraindicated in somesituations due to their tendency to induce ketogenesis and metabolicacidosis. Due to their ability to be absorbed rapidly by the body,medium-chain triglycerides have found use in the treatment of a varietyof malabsorption ailments. MCT supplementation with a low-fat diet hasbeen described as the cornerstone of treatment for primary intestinallymphangiectasia (Waldmann's disease). MCTs are an ingredient inparenteral nutritional emulsions. Accordingly, in some embodiments, thexylose compositions are capable of increasing the level of one or moremedium chain triglycerides in a mammalian subject. In certainembodiments, the administration of the xylose composition to a mammaliansubject results in the increase of one or more medium chaintriglycerides in the mammalian subject by approximately 1.5-fold,2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater than100-fold.

Distal disorders associated with loss of intestinal barrier function canbe treated or improved by administration of probiotic compositionscontaining bacterial strains that produce short chain fatty acids(SCFAs), such as, for example, butyrate, acetate, propionate, orvalerate, or combinations thereof. Distal disorders associated with lossof intestinal barrier function can be treated or improved byadministration of probiotic compositions containing bacterial strainsthat reduce inflammation, as described herein.

In other embodiments, the distal dysbiosis is caused by a deficiency inmicrobes that produce lactic acid. Accordingly, in one embodiment, theprobiotic composition can contain a species of bacteria that producelactic acid.

Probiotic compositions for modulating a distal microbiome may optionallybe administered in conjunction with a prebiotic. For example, aprebiotic can be selected which augments the growth of theanti-inflammatory bacterial population present in the probioticcomposition. Exemplary prebiotics are provided in Table 7. Exemplaryprebiotics which may augment the growth of exemplary bacterial speciesare provided in FIG. 29. In one embodiment, the prebiotic can be amonomer or polymer selected from the group consisting of arabinoxylan,xylose, soluble fiber dextran, soluble corn fiber, polydextrose,lactose, N-acetyl-lactosamine, glucose, or combinations thereof. Inanother embodiment, the prebiotic can be a monomer or polymer, such asgalactose, fructose, rhamnose, mannose, uronic acids, 3′-fucosyllactose,3′sialylactose, 6′-sialyllactose, lacto-N-neotetraose,2′-2′-fucosyllactose, or combinations thereof. In one embodiment, theprebiotic can include a monosaccharide selected from the groupconsisting of arabinose, fructose, fucose, lactose, galactose, glucose,mannose, D-xylose, xylitol, ribose, and combinations thereof. In anotherembodiment, the prebiotic can include a disaccharide selected from thegroup consisting of xylobiose, sucrose, maltose, lactose, lactulose,trehalose, cellobiose, or a combination thereof. In another embodiment,the prebiotic comprises a polysaccharide, for example, axylooligosaccharide. Exemplary prebiotics include sugars such asarabinose, fructose, fucose, lactose, galactose, glucose, mannose,D-xylose, xylitol, ribose, xylobiose, sucrose, maltose, lactose,lactulose, trehalose, cellobiose, and xylooligosaccharide, orcombinations thereof.

The foregoing probiotic compositions (and optional prebioticcompositions) can be used for treatment of the following disordersassociated with dysbiosis of the microbiome at particular niches withinthe subject, or with disorders of the systemic microbiome.

IX. Autoimmune/Inflammatory Diseases

Herein, we disclose probiotic microbial compositions, optionallycomprising prebiotics, non-microbial immunomodulatory carbohydrates, ormicrobial immunomodulatory cell components, that are effective for theprevention or treatment of transplant disorders in a transplantrecipient. Such disorders, e.g., GVHD, transplant rejection, sepsis,etc. are associated with systemic inflammation and/or loss of intestinalbarrier function. In one embodiment, the transplant recipient has anautoimmune or inflammatory disorder. For example, a subject with anautoimmune or inflammatory disorder may receive a transplant, e.g., ahematopoietic stem cell transplant, for example, and autologoushematopoietic stem cell transplant, as a treatment modality for theautoimmune or inflammatory disorder. In this embodiment, administrationof the probiotic (and optional prebiotic) compositions of the inventioncan be used to prevent or treat GVHD in the subject receiving thetransplant, and can additionally or alternatively be used to treat theunderlying autoimmune or inflammatory disorder. Exemplary autoimmune orinflammatory disorders include, for example, lupus, multiple sclerosis,systemic sclerosis, Crohn's disease, type I diabetes, or juvenileidiopathic arthritis. Additional autoimmune or inflammatory disordersinclude, for example, an inflammatory bowel disease (IBD) including butnot limited to ulterative colitis and Crohn's disease, multiplesclerosis (MS), systemic lupus erythematosus (SLE), type I diabetes,rheumatoid arthritis, Sjögren's syndrome, and Celiac disease. In certainembodiments, the compositions comprise at least one type of microbe andat least one type of carbohydrate (a prebiotic), and optionally furthercomprise microbial immunomodulatory cell components or substrates forthe production of immunomodulatory metabolites, that are effective forthe prevention or treatment of an autoimmune or inflammatory disorder.We also disclose herein methods for the prevention and/or treatment ofautoimmune and inflammatory diseases in human subjects, e.g., transplantrecipients.

In one embodiment, the subject is receiving a hematopoietic stem celltransplant. In other embodiments, the subject is receiving a bone marrowtransplant. In other embodiments, the subject is receiving a solid organtransplant, e.g., a kidney transplant, a heart transplant, a lungtransplant, a skin transplant, a liver transplant, a pancreastransplant, an intestinal transplant, an endocrine gland transplant, abladder transplant, and/or a skeletal muscle transplant.

Autoimmune and inflammatory diseases include, but are not limited to:Acute Disseminated Encephalomyelitis, Acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, adhesive capsulitis,Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Anti-GBM nephritis, Anti-TBM nephritis, Antiphospholipidsyndrome, arthofibrosis, atrial fibrosis, autoimmune angioedema,autoimmune aplastic anemia, autoimmune dusautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease, autoimmune myocarditis, autoimmuneoophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmunethrombocytopenic purpura, autoimmune thyroid disease, autoimmuneurticaria, axonal and neuronal neuropathies, Balo disease, Behçet'sdisease, benign mucosal pemphigold, Bullous pemphigold, cardiomyopathy,Castleman disease, Celiac Disease, Chagas disease, chronic fatiguesyndrome, chronic inflammatory demyelinating polyneuropathy, chronicLyme disease, chronic recurrent multifocal osteomyelitis, Churg-Strausssyndrome, cicatricial pemphigold, cirrhosis, Cogans syndrome, coldagglutinin disease, congenital heart block, Coxsackle myocarditis, CRESTdisease, Crohn's disease, Cystic Fibrosis, essential mixedcryoglobulinemia, deficiency of the interleukin-1 receptor antagonist,demyelinating neuropathies, dermatitis herpetiformis, dermatomyosis,Devic's disease, discoid lupus, Dressler's syndrome, Dupuytren'scontracture, endometriosis, endomyocardial fibrosis, eosinophilicesophagitis, eosinophilic facsciitis, erythema nodosum, experimentalallergic encephalomyelitis, Evans syndrome, Familial MediterraneanFever, fibromyalgia, fibrosing alveolitis, giant cell arteritis, giantcell myocarditis, glomerulonephritis, Goodpasture's syndrome,Graft-versus-host disease (GVHD), granulomatosus with polyanglitis,Graves' disease, Guillain-Bare syndrome, Hashimoto's encephalitis,Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura,hepatitis, herpes gestationis, hypogammaglobulinemia, idiopathicthrombocytopenic purpura, IgA nephropathy, IgG4-related sclerosingdisease, immunoregulatory lipoproteins, inclusion body myositis,inflammatory bowel disorders, interstitial cystitis, juvenile arthritis,juvenile myositis, Kawasaki syndrome, keloid, Lambert-Eaton syndrome,leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneousconjunctivitis, linear IgA disease, mediastinal fibrosis, Meniere'sdisease, microscopic polyanglitis, mixed connective tissue disease,Mooren's ulcer, Mucha-Hamermann disease, Multiple Sclerosis (MS),Myasthenia gravis, myelofibrosis, Myositis, narcolepsy, Neonatal OnsetMultisystem Inflammatory Disease, nephrogenic systemic fibrosis,neutropenia, nonalcoholic fatty liver disease, nonalcoholicsteatohepatitis (NASH), ocular-cicatricial pemphigold, optic neuritis,palindromic rheumatism, Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus (PANDAS), paraneoplastic cerebellardegeneration, paroxysmal nocturnal nemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia,Peyronie's disease, POEMS syndrome, polyarteritis nodosa, progressivemassive fibrosis, Tumor Necrosis Factor Receptor-assoicated PeriodicSyndrome, Type I autoimmune polyglandular syndrome, Type II autoimmunepolyglandular syndrome, Type III autoimmune polyglandular syndrome,polymyalgia rhematica, polymyositis, postmyocardial infarction syndrome,postpericardiotomy syndrome, progesterone dermatitis, primary biliarycirrhosis, primary sclerosing cholangitis, psoriasis, psoriaticarthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure redcell aplasia, Raynauds phenomenon, reactic arthritis, reflex sympatheticdystrophy, Reiter's syndrome, relapsing polychondritis, restless legssyndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoidarthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma,Sjögren's syndrome, sperm and testicular autoimmunity, stiff personsyndrome, subacute bacterial endocarditis, Susac's syndrome, sympatheticophthalmia, systemic lupus erythematosus (SLE), Takayasu's arthritis,temporal arteritis, thrombocytopenic purpura, Tolosa-Hunt syndrome,transverse myelitis, Type 1 diabetes, ulcerative colitis,undifferentiated connective tissue disease, uveitis, vasculitis,vesiculobullous dermatosis, and Vitiligo.

In some aspects, the administered microbes and/or carbohydrates modulatethe release of immune stimulatory cytokines. In preferred embodiments,the administered microbes and/or carbohydrates inhibit or reduce therelease of immune stimulatory cytokines. Non-limiting examples of immunemodulating cytokines and ligands include B lymphocyte chemoattractant(“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotacticprotein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor(“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”),1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma(“IFN-γ”), Interlukin-1 alpha (“IL-1α”), Interlukin-113 (“IL-1”),Interleukin 1 receptor antagonist (“IL-1 ra”), Interleukin-2 (“IL-2”),Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”),Interleukin-6 soluble receptor (“IL-6 sR”), Interleukin-7 (“IL-7”),Interleukin-8 (“IL-8”), Interleukin-10 (“IL-10”), Interleukin-11(“IL-11”), Subunit β of Interleukin-12 (“IL-12 p40” or “IL-12 p70”),Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16(“IL-16”), Interleukin-17 (“IL-17”), Chemokine (C-C motif) Ligand 2(“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokineinduced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2(“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1β”), Macrophageinflammatory protein-1-δ (“MIP-1δ”), Platelet-derived growth factorsubunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated onActivation, Normal T cell Expressed and Secreted (“RANTES”), TIMPmetallopeptidase inhibitor 1 (“TIMP-1”), TIMP metallopeptidase inhibitor2 (“TIMP-2”), Tumor necrosis factor, lymphotoxin-α (“TNF-α”), Tumornecrosis factor, lymphotoxin-β (“TNF β”), Soluble TNF receptor type 1(“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basicfibroblast growth factor (“bFGF”), Bone morphogenetic protein 4(“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogeneticprotein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growthfactor (“EGF”), Epidermal growth factor receptor (“EGFR”),Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”),Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor(“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glialcell-derived neurotrophic factor (“GDNF”), Growth Hormone,Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growthfactor (“HGF”), Insulin-like growth factor binding protein 1(“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”),Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-likegrowth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factorbinding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”),Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growthfactor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4(“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”),Platelet-derived growth factor receptors (“PDGF-AA”),Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-boxcontaining comples (“SCF”), Stem cell factor receptor (“SCF R”),Transforming growth factor α (“TGF-α”), Transforming growth factor β-1(“TGF β1”), Transforming growth factor β-3 (“TGF β3”), Vascularendothelial growth factor (“VEGF”), Vascular endothelial growth factorreceptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3(“VEGFR3”), VEGF-D 6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”),Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”),Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif)ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA-78”), Chemokine (C-Cmotif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2(“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-1”), Chemokine(C-C motif) ligand 16 (“HCC-4”), Interleukin-9 (“IL-9”), Interleukin-17F (“IL-17F”), Interleukin-18-binding protein (“IL-18 BPa”),Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31(“IL-31”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3(“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (Cmotif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2(“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocytechemoattractant protein 4 (“MCP-4”), Macrophage-derived chemokine(“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-Cmotif) ligand 20 (“MIP-3 α”), C-C motif chemokine 19 (“MIP-3 (3”),Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulatingprotein alpha chain (“MSP-α”), Nucleosome assembly protein 1-like 4(“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary andactivation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”),Stroma cell-derived factor-1α (“SDF-1 α”), Chemokine (C-C motif) ligand17 (“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromallymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster ofDifferentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamilymember 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster ofDifferentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1(biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein(“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”),Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosisantigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosisfactor receptor superfamily member 1 (“GITR”), Tumor necrosis factorreceptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rβ, IL-17R, IL-2Rγ, IL-21R,Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associatedlipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium(“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHCclass I polypeptide-related sequence B (“MICB”), NRGl-131, Beta-typeplatelet-derived growth factor receptor (“PDGF Rβ”), Plateletendothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A viruscellular receptor 1 (“TIM-1”), Tumor necrosis factor receptorsuperfamily member IOC (“TRAIL R3”), Trappin protein transglutaminasebinding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular celladhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein(“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin,Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN,Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial celladhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C,FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”),IL-13 Rl, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal celladhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1(“PAI-I”), Platelet derived growth factor receptors (“PDGF-AB”),Resistin, stromal cell-derived factor 1 (“SDF-1 β”), sgpl30, Secretedfrizzled-related protein 2 (“ShhN”), Sialic acid-bindingimmunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growthfactor-β 2 (“TGF β 2”), Tie-2, Thrombopoietin (“TPO”), Tumor necrosisfactor receptor superfamily member 10D (“TRAIL R4”), Triggering receptorexpressed on myeloid cells 1 (“TREM-1”), Vascular endothelial growthfactor C (“VEGF-C”), VEGFRlAdiponectin, Adipsin (“AND”), α-fetoprotein(“AFP”), Angiopoietin-like 4 (“ANGPTL4”), β-2-microglobulin (“B2M”),Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125(“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen(“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth FactorReceptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”),Chemokine (C-X-C motif) ligand 1 (“GRO α”), human chorionic gonadotropin(“β HCG”), Insulin-like growth factor 1 receptor (“IGF-1 sR”), IL-1sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase-1(“MMP-1”), Matrix metalloproteinase-2 (“MMP-2”), Matrixmetalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”),Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase-10(“MMP-10”), Matrix metalloproteinase-13 (“MMP-13”), Neural Cell AdhesionMolecule (“NCAM-1”), Entactin (“Nidogen-1”), Neuron specific enolase(“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostatespecific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9(“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin,Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin andmetalloproteinase domain-containing protein 9 (“ADAM-9”), Angiopoietin2, Tumor necrosis factor ligand superfamily member 13/Acidicleucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bonemorphogenetic protein 2 (“BMP-2”), Bone morphogenetic protein 9(“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97,Chemerin, Tumor necrosis factor receptor superfamily member 6B (“DcR3”),Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein,alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3,Hepatocyte growth factor receptor (“HGF R”), IFN-ya/3 R2, Insulin-likegrowth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF-2R”), Interleukin-1 receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”),Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase(“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-1”),Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1,translocation-associated (Drosophila) (“Notch-1”), Nephroblastomaoverexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1(“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4,Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin,Toll-like receptor 2 (“TLR2”), Tumor necrosis factor receptorsuperfamily member 10A (“TRAIL Rl”), Transferrin (“TRF”), WIF-1ACE-2,Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”),Carbohydrate antigen 19-9 (“CA19-9”), CD 163, Clusterin, CRT AM,Chemokine (C-X-C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin(“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Delta-like protein 1(“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folatereceptor α (“FOLR”), Furin, GPCR-associated sorting protein 1(“GASP-1”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocytecolony-stimulating factor receptor (“GCSF R”), Serine protease hepsin(“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27(“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V(“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST,Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factorreceptor superfamily member 13B (“TACI”), Tissue factor pathwayinhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily,member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase PlasminogenActivator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascularendothelial) also known as CD 144 (“VE-Cadherin”),WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and ReceptorActivator of Nuclear Factor κ B (“RANK”).

Exemplary probiotic compositions useful for treatment or prevention ofautoimmune or inflammatory disorders contain bacterial strains capableof reducing inflammation in a subject. Such immunomodulatory(anti-inflammatory) bacteria can modulate cytokine expression by hostimmune cells, resulting in an overall increase in secretion ofanti-inflammatory cytokines and/or an overall decrease in secretion ofpro-inflammatory cytokines, systemically reducing inflammation in thesubject. In exemplary embodiments, probiotic compositions useful fortreatment of immune or inflammatory disorders stimulate secretion of oneor more anti-inflammatory cytokines by host immune cells, such as PBMCs.Anti-inflammatory cytokines include, but are not limited to, IL-10,IL-13, IL-9, IL-4, IL-5, TGFβ and combinations thereof. In otherexemplary embodiments, probiotic compositions useful for treatment ofautoimmune or inflammatory disorders inhibit secretion of one or morepro-inflammatory cytokines by host immune cells, such as PBMCs.Pro-inflammatory cytokines include, but are not limited to, IFNγ,IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinationsthereof. Other exemplary cytokines are known in the art and aredescribed herein. Probiotic compositions containing anti-inflammatorybacteria reduce inflammation at the site of administration, e.g., in thegastrointestinal tract, as well as at distal sites throughout the bodyof the subject.

Other exemplary probiotic compositions useful for treatment ofautoimmune or inflammatory disorders contain bacterial strains capableof altering the proportion of immune subpopulations, e.g., T cellsubpopulations, in the subject.

For example, immunomodulatory bacteria can increase or decrease theproportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in asubject. The increase or decrease in the proportion of immune cellsubpopulations may be systemic, or it may be localized to a site ofaction of the probiotic, e.g., in the gastrointestinal tract or at thesite of a distal dysbiosis. In some embodiments, a probiotic compositioncomprising immunomodulatory bacteria is used for treatment of anautoimmune or inflammatory disorder based on the desired effect of theprobiotic composition on the differentiation and/or expansion ofsubpopulations of immune cells in the subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria that increase the proportion of Treg cells in a subject. Inanother embodiment, a probiotic composition contains immunomodulatorybacteria that decrease the proportion of Treg cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th17 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th17 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th1 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th1 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th2 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th2 cells in a subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria capable of modulating the proportion of one or more of Tregcells, Th17 cells, Th1 cells, and combinations thereof in a subject.Certain immune cell profiles may be particularly desirable to treat orprevent autoimmune or inflammatory disorders. For example, in someembodiments, treatment or prevention of autoimmune or inflammatorydisorders can be promoted by increasing numbers of Treg cells and Th2cells, and decreasing numbers of Th17 cells and Th1 cells. Accordingly,probiotic compositions for the treatment or prevention of autoimmune orinflammatory disorders may contain probiotics capable of promoting Tregcells and Th2 cells, and reducing Th17 and Th1 cells.

Probiotic compositions useful for treating or preventing the autoimmuneor inflammatory disorders described herein include, in exemplaryembodiments, one or more bacterial strains from Table 1. In otherembodiments, the probiotic composition includes one or more bacterialstrains from Table 1A. In other embodiments, the probiotic compositionincludes one or more bacterial strains from Table 1B. In otherembodiments, the probiotic composition includes one or more bacterialstrains from Table 1C. In other embodiments, the probiotic compositionincludes one or more bacterial strains from Table 1D. In otherembodiments, the probiotic composition includes one or more bacterialstrains from Table 1E. In other embodiments, the probiotic compositionincludes one or more bacterial strains from Table 1F. In someembodiments, the probiotic composition contains a single strain ofbacteria. In other embodiments, the probiotic composition contains twoor more strains of bacteria, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more strains of bacteria.In other embodiments, the probiotic composition contains or isadministered in conjunction with a prebiotic, as described herein.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter.

Preferred bacterial genera also include Acetonema, Alkaliphilus,Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor,Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella,Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Halobacteroides, Heliobacterium, Heliophilum, Heliorestis,Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.),Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora,Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum,Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus,Thermoanaerobacter, and Thermosinus.

As provided herein, therapeutic compositions comprise, or in thealternative, modulate, the colonization and/or engraftment, of thefollowing exemplary bacterial entities: Lactobacillus gasseri,Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis,Enterococcus durans, Enterococcus villorum, Lactobacillus plantarum,Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcuscohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcusmitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcusanginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridiumsaccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6,Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcusgnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautiaproducta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonellaparvula, Clostridium methylpentosum, Clostridium islandicum,Faecalibacterium prausnitzii, Bacteroides uniformmis, Bacteroidesthetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus,Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirumpropionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria,Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta.

Preferred bacterial species are provided in Table 1, Table 1A, Table 1B,Table 1C, Table 1D, Table 1E, Table 1F, and Table 5. Optionally, in someembodiments, preferred bacterial species are spore formers. Wherespecific strains of a species are provided, one of skill in the art willrecognize that other strains of the species can be substituted for thenamed strain.

In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Acidaminococcusintestine. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isAcinetobacter baumannii. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Acinetobacter lwoffii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Akkermansia muciniphila. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Alistipes putredinis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Alistipes shahii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerostipes hadrus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerotruncus colihominis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides caccae.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidescellulosilyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isBacteroides dorei. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides eggerthii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides finegoldii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides fragilis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroides massiliensis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides ovatus.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidessalanitronis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBacteroides salyersiae. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides sp. 1_1_6. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides sp. 3_1_23. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides sp. D20. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroidesthetaiotaomicrond. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides uniformis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides vulgatus. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bifidobacterium adolescentis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacterium bifidum. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Bifidobacterium breve.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bifidobacteriumfaecale. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBifidobacterium kashiwanohense. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Bifidobacterium longum subsp. Longum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bifidobacterium stercoris. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Blautia (Ruminococcus) coccoides. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia faecis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia glucerasea. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)hansenii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)luti. In one embodiment, the bacterial entity, e.g., species or strain,useful in the compositions and methods of the invention is Blautia(Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia producta (Ruminococcus productus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)schinkii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia stercoris. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1).In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Blautia unculturedbacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia wexlerae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Candidatus Arthromitus sp.SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isCatenibacterium mitsuokai. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridiales bacterium1_7_47FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium asparagiforme. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridium bolteae. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium clostridioforme. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium glycyrrhizinilyticum. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium (Hungatella)hathewayi. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium histolyticum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium indolis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium leptum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium (Tyzzerella) nexile. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium perfringens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium(Erysipelatoclostridium) ramosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium scindens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium septum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium sp. 14774. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium sp.7_3_54FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium sp. HGF2. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium symbiosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Collinsella aerofaciens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Collinsella intestinalis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Coprobacillus sp. D7. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus catus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus comes. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Dorea formicigenerans.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Dorea longicatena.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Enterococcusfaecalis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEnterococcus faecium. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli S88. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacterium eligens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacteriumfissicatena. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEubacterium ramulus. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Eubacterium rectale. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Faecalibacterium prausnitzii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Flavonifractor plautii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Fusobacterium mortiferum.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Fusobacteriumnucleatum. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isHoldemania filiformis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Hydrogenoanaerobacterium saccharovorans. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Klebsiella oxytoca. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isLachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Lachnospiraceae bacterium 5_1_57FAA. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus casei. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus rhamnosus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Lactobacillus ruminis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Lactococcus casei.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Odoribactersplanchnicus. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isOscillibacter valericigenes. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Parabacteroides gordonii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Parabacteroides johnsonii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Parabacteroides merdae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Pediococcus acidilactici.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Peptostreptococcusasaccharolyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isPropionibacterium granulosum. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Roseburia intestinalis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Roseburia inulinivorans. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Ruminococcus faecis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus gnavus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus sp. ID8. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Ruminococcus torques.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Slackia piriformis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Staphylococcusepidermidis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStaphylococcus saprophyticus. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Streptococcus cristatus. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus dysgalactiae subsp. Equisimilis. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Streptococcus infantis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Streptococcusoralis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStreptococcus sanguinis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Streptococcus viridans. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus thermophiles. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Veillonella dispar.

In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Acidaminococcus intestine. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter baumannii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter lwoffii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Akkermansia muciniphila. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes putredinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes shahii. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Anaerostipes hadrus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Anaerotruncus colihominis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Bacteroides caccae. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides cellulosilyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides dorei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides eggerthii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides finegoldii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides fragilis. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Bacteroidesmassiliensis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Bacteroides ovatus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bacteroides salanitronis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides salyersiae. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 1_1_6. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 3_1_23. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. D20. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides thetaiotaomicrond. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides uniformis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides vulgatus. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium adolescentis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium bifidum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium breve. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium faecale. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium kashiwanohense. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium longum subsp. Longum.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Bifidobacteriumstercoris. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) coccoides. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia faecis. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiaglucerasea. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) hansenii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) luti. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) obeum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia producta (Ruminococcusproductus). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) schinkii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia stercoris. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiauncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia uncultured bacterium cloneSJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia wexlerae. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Catenibacterium mitsuokai. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridiaceae bacterium (Dielmafastidiosa) JC13. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Clostridialesbacterium 1_7_47FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumasparagiforme. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium bolteae.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium clostridioforme. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium glycyrrhizinilyticum. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Hungatella) hathewayi.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium histolyticum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium indolis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium leptum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Tyzzerella) nexile. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium perfringens. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Erysipelatoclostridium)ramosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumscindens. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium septum. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumsp. 14774. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.7_3_54FAA. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.HGF2. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumsymbiosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaaerofaciens. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaintestinalis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprobacillus sp.D7. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprococcus catus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Coprococcus comes. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea formicigenerans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea longicatena. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecalis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecium. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Erysipelotrichaceae bacterium 3_1_53. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Escherichia coli. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Escherichia coli S88. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium eligens. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium fissicatena. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium ramulus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium rectale. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Faecalibacterium prausnitzii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Flavonifractor plautii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium mortiferum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium nucleatum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Holdemania filiformis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Hydrogenoanaerobacterium saccharovorans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Klebsiella oxytoca. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Lachnospiraceaebacterium 7_1_58FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprisesLachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus casei. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus rhamnosus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus ruminis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactococcus casei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOdoribacter splanchnicus. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOscillibacter valericigenes. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides gordonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides johnsonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides merdae. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPediococcus acidilactici. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPeptostreptococcus asaccharolyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Propionibacterium granulosum. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia intestinalis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia inulinivorans. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus faecis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus gnavus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus sp. ID8. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus torques. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Slackia piriformis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus epidermidis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus saprophyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus cristatus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus dysgalactiae subsp. Equisimilis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus infantis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus oralis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus sanguinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus viridans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus thermophiles. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Veillonella dispar.

In one embodiment, the prebiotic carbohydrate component of thepharmaceutical composition, dosage form, or kit allows the commensalcolonic microbiota, comprising microorganisms associated with ahealthy-state microbiome or presenting a low risk of a patientdeveloping an autoimmune or inflammatory condition, to be regularlymaintained. In one embodiment, the prebiotic carbohydrate component ofthe pharmaceutical composition, dosage form, or kit allows theco-administered or co-formulated microbe or microbes to engraft, grow,and/or be regularly maintained in a mammalian subject. In someembodiments, the mammalian subject is a human subject. In preferredembodiments, the mammalian subject suffers from or is at risk ofdeveloping an autoimmune or inflammatory disorder. In some embodiments,the prebiotic component of the invention favors the growth of anadministered microbe, wherein the growth of the administered microbeand/or the fermentation of the administered prebiotic by theadministered microbe slows or reduces the growth of a pathogen orpathobiont. For example, FOS, neosugar, or inuliri promotes the growthof acid-forming bacteria in the colon such as bacteria belonging to thegenera Lactobacillus or Bifidobacterium and Lactobacillus acidophilusand Bifidobacterium bifidus can play a role in reducing the number ofpathogenic bacteria in the colon (U.S. Pat. No. 8,486,668 PREBIOTICFORMULATIONS AND METHODS OF USE). Other polymers, such as variousgalactans, lactulose, and carbohydrate based gums, such as psyllium,guar, carrageen, gellan, and konjac, are also known to improvegastrointestinal (GI) health.

Short chain fatty acids (SCFAs) can have immunomodulatory (i.e.,immunosuppressive) effects and therefore their production (i.e.,biosynthesis or conversion by fermentation) is advantageous for theprevention, control, mitigation, and treatment of autoimmune and/orinflammatory disorders (Lara-Villoslada F. et al., 2006. Short-chainfructooligosaccharides, in spite of being fermented in the upper part ofthe large intestine, have anti-inflammatory activity in the TNBS modelof colitis. Eur J Nutr. 45(7): 418-425). In some aspects, thepharmaceutical composition, dosage form, or kit comprises at least onetype of microbe and at least one type of prebiotic such that thecomposition, dosage form, or kit is capable of increasing the level ofone or more immunomodulatory SCFA (e.g., acetate, propionate, butyrate,or valerate) in a mammalian subject. Optionally, the pharmaceuticalcomposition, dosage form, or kit further comprises one or moresubstrates of one or more SCFA-producing fermentation and/orbiosynthesis pathways. In certain embodiments, the administration of thecomposition, dosage form, or kit to a mammalian subject results in theincrease of one or more SCFAs in the mammalian subject by approximately1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, orgreater than 100-fold.

In some embodiments, the prebiotic mixture is selected to favor theproduction of a particular immunomodulatory SCFA, including but notlimited to butyrate, propionate, or acetate. In preferred embodiments,the fermentation product is butyrate or propionate. Non-limitingexamples are resistant starch and carbohydrates with highly organizedstructures, such as high amylose maize starch, that are more likely tobe fermented by microbes to produce butyrate than other SCFAs (Zhou Z etal. 2013. Starch structure modulates metabolic activity and gutmicrobiota profile. Anaerobe. 24:71-78). In some embodiments, one ormore components of a prebiotic mixture is subjected to denaturation(e.g., thermal treatment) to favor the production of a SCFA (e.g.,acetate) or a non-SCFA species including but not limited to lactate orsuccinate.

In some embodiments, the pharmaceutical composition, dosage form, or kitcomprises one or more types of microbe capable of producing butyrate ina mammalian subject. Butyrate-producing microbes may be identifiedexperimentally, such as by NMR or gas chromatography analyses ofmicrobial products or colorimetric assays (Rose I A. 1955. MethodsEnzymol. Acetate kinase of bacteria. 1: 591-5). Butyrate-producingmicrobes may also be identified computationally, such as by theidentification of one or more enzymes involved in butyrate synthesis.Non-limiting examples of enzymes found in butyrate-producing microbesinclude butyrate kinase, phosphotransbutyrylase, and butyryl CoA:acetateCoA transferase (Louis P., et al. 2004. Restricted Distribution of theButyrate Kinase Pathway among Butyrate-Producing Bacteria from the HumanColon. J Bact. 186(7): 2099-2106). Butyrate-producing strains include,but are not limited to, Faecalibacterium prausnitzii, Eubacterium spp.,Butyrivibrio fibrisolvens, Roseburia intestinalis, Clostridium spp.,Anaerostipes caccae, and Ruminococcus spp. In some embodiments, thepharmaceutical composition, dosage form, or kit comprises two or moretypes of microbe, wherein at least two types of microbe are capable ofproducing butyrate in a mammalian subject. In other embodiments, thepharmaceutical composition, dosage form, or kit comprises two or moretypes of microbe, wherein two or more types of microbe cooperate (i.e.,cross-feed) to produce an immunomodulatory SCFA (e.g., butyrate) in amammalian subject. In a preferred embodiment, the pharmaceuticalcomposition, dosage form, or kit comprises at least one type of microbe(e.g., Bifidobacterium spp.) capable of metabolizing a prebiotic,including but not limited to, inulin, inulin-type fructans, oroligofructose, such that the resulting metabolic product may beconverted by a second type of microbe (e.g, a butyrate-producing microbesuch as Roseburia spp.) to an immunomodulatory SCFA such as butyrate(Falony G., et al. 2006. Cross-Feeding between Bifidobacterium longumBB536 and Acetate-Converting, Butyrate-Producing Colon Bacteria duringGrown on Oligofructose. Appl. Environ. Microbiol. 72(12): 7835-7841.) Inother aspects, the pharmaceutical composition, dosage form, or kitcomprises at least one acetate-producing microbe (e.g., Bacteroidesthetaiotaomicron) and at least one acetate-consuming, butyrate-producingmicrobe (e.g., Faecalibacterium prausnitzii).

In some embodiments, the pharmaceutical composition, dosage form, or kitcomprises one or more types of microbe capable of producing propionatein a mammalian subject, optionally further comprising a prebiotic orsubstrate appropriate for proprionate biosynthesis. Examples ofprebiotics or substrates used for the production of propionate include,but are not limited to, L-rhamnose, D-tagalose, resistant starch,inulin, polydextrose, arabinoxylans, arabinoxylan oligosaccharides,mannooligosaccharides, and laminarans (Hosseini E., et al. 2011.Propionate as a health-promoting microbial metabolite in the human gut.Nutrition Reviews. 69(5): 245-258). Propionate-producing microbes may beidentified experimentally, such as by NMR or gas chromatography analysesof microbial products or colorimetric assays (Rose I A. 1955. MethodsEnzymol. Acetate kinase of bacteria. 1: 591-5). Propionate-producingmicrobes may also be identified computationally, such as by theidentification of one or more enzymes involved in propionate synthesis.Non-limiting examples of enzymes found in propionate-producing microbesinclude enzymes of the succinate pathway, including but not limited tophophoenylpyrvate carboxykinase, pyruvate kinase, pyruvate carboxylase,malate dehydrogenase, fumarate hydratase, succinate dehydrogenase,succinyl CoA synthetase, methylmalonyl Coa decarboxylase, and propionateCoA transferase, as well as enzymes of the acrylate pathway, includingbut not limited to L-lactate dehydrogenase, propionate CoA transferase,lactoyl CoA dehydratase, acyl CoA dehydrogenase, phosphateacetyltransferase, and propionate kinase. Non-limiting examples ofmicrobes that utilize the succinate pathway are Bacteroides fragilis andother species (including B. vulgatus), Propionibacterium spp. (includingfreudenrichii and acidipropionici), Veillonella spp. (includinggazogenes), Micrococcus lactilyticus, Selenomonas ruminantium,Escherichia coli, and Prevotella ruminocola. Non-limiting examples ofmicrobes that utilize the acrylate pathway are Clostridiumneopropionicum X4, and Megasphaera elsdenii. In preferred embodiments,the combination of a type of microbe or microbial composition and typeof prebiotic mixture is selected based on the fermentation or metabolicpreferences of one or more microbes capable of producingimmunomodulatory SCFAs (e.g., preference for complex versus simple sugaror preference for a fermentation product versus a prebiotic). Forexample, M. eldsenii prefers lactate fermentation to glucosefermentation, and maximization of propionate production by M. eldseniiin a mammalian subject may therefore be achieved by administering alongwith M. eldsenii a favored substrate (e.g., lactate) or one or moremicrobes capable of fermenting glucose into lactate (e.g., Streptococcusbovis) (Hosseini E., et al. 2011. Propionate as a health-promotingmicrobial metabolite in the human gut. Nutrition Reviews. 69(5):245-258). Thus, in some embodiments, the pharmaceutical composition,dosage form, or kit comprises at least one type of SCFA-producingmicrobe and a sugar fermentation product (e.g., lactate). In otherembodiments, the pharmaceutical composition, dosage form, or kitcomprises at least one type of SCFA-producing microbe and at least onetype of sugar-fermenting microbe, wherein the fermentation product ofthe second, sugar-fermenting microbe is the preferred substrate of theSCFA-producing microbe.

In some embodiments, the pharmaceutical composition, dosage form, or kitcomprises two or more types of microbe, wherein at least two types ofmicrobe are capable of producing propionate in a mammalian subject. Inother embodiments, the pharmaceutical composition, dosage form, or kitcomprises two or more types of microbe, wherein two or more types ofmicrobe cooperate (i.e., cross-feed) to produce an immunomodulatory SCFA(e.g., propionate) in a mammalian subject. In a preferred embodiment,the pharmaceutical composition, dosage form, or kit comprises at leastone type of microbe (e.g., Ruminococcus spp. or Bacteroides spp.)capable of metabolizing a prebiotic into succinate, and a second type ofmicrobe (e.g., S. ruminantium) capable of converting succinate (via thesuccinate pathway) into propionate in the mammalian subject.

Immunomodulation can also be achieved by the microbial production ofglutathione or gamma-glutamylcysteine. Thus, in certain embodiments, thepharmaceutical composition, dosage form, or kit comprises at least onetype of microbe capable of producing glutathione and/orgamma-glutamylcysteine in a mammalian subject. In some aspects, thecomposition, dosage form, or kit comprises one or more microbes selectedfor the presence of glutamate cysteine ligase (e.g., Lactobacillusfermentum) and/or L-proline biosynthesis enzymes (e.g., E. coli) (Peranet al., 2006. Lactobacillus fermenum, a probiotic capable to releaseglutathione, prevents colonic inflammation in the TNBS model of ratcolitis. Int J Colorectal Dis. 21(8): 737-746; Veeravalli et al., 2011.Laboratory evolution of glutathione biosynthesis reveals naturallycompensatory pathways. Nat Chem Bio. 7(2): 101-105). In a preferredembodiment, at least one microbe in the pharmaceutical composition,dosage form, or kit is L. fermentum.

para-cresol (p-cresol) is a microbial product, via the fermentation oftyrosine or phenylalanine. Sulfated in the liver or colon to p-cresylsulfate, this molecule reduces Th1-mediated responses (Shiba T. et al.2014. Effects of intestinal bacteria-derived p-cresyl sulfate onTh1-type immune response in vivo and in vitro. Tox and Applied Pharm.274(2): 191-199). In some embodiments, the pharmaceutical composition,dosage form, or kit comprises at least one type of microbe capable offermenting tyrosine and/or phenylalanine to p-cresol in a mammaliansubject. Non-limiting examples of such microbes include Bacteroidesfragilis, Clostridium difficile, and Lactobacillus sp. Strain#11198-11201 (Yokoyama M T and Carlson J R. 1981. Production of Skatoleand para-Cresol by a Rumen Lactobacillus sp. Applied and EnvironmentalMicrobiology. 41(1): 71-76.), and other microbes with p-hydroxylphenylacetate decarboxylase activity.

It has recently come to light that the DNA of commensal microbes,including many species of Lactobacillus protect against activation oflamina propia dendritic cells and sustain regulatory T cell conversion(Bouladoux N, Hall J A, Grainger J R, dos Santos L M, Kann M G,Nagarajan V, Verthelyi D, and Belkaid Y, 2012. Regulatory role ofsuppressive motifs from commensal DNA. Mucosal Immunol. 5: 623-634).Thus commensal DNA may protect against colitis, IBD, and/or otherimmunological intolerances in the gut. Furthermore, Lactobacillusspecies are prevalent in the healthy vaginal microbiome. Thus, DNA fromLactobacillus or other vaginal microbiome commensals may suppress immuneresponses in the vagina that could disrupt the normal healthy-statevaginal microbiome and lead to complications such as chronic HPV,infertility, miscarriages, or UTIs. As such, in certain embodiments, themicrobial composition, pharmaceutical composition, dosage form, or kitadditionally comprises DNA isolated from one or more host commensals.

X. Crohn's Disease

Crohn's disease and ulcerative colitis are types of IBDs. While bothillness share elements of their characteristic immune responses (e.g.,high TNF-α, which can be detected in a patient's feces), theirassociated immune responses can also have distinguishing markers. Forexample, interleukin-16 (IL-16) levels are high and T-bet isoverexpressed the lamina propia T cell nucleus in patients with Crohn'sdisease, but not in those suffering from ulcerative colitis. NotablyT-bets produce the pro-inflammatory cytokine IFN-γ. One similarity amongIBDs is high IFN-γ (by about 4-fold), caused in part due to high TL1Aand TNF-α. Moreover, the levels of these cytokines correlate with theseverity of the IBD.

Early Crohn's disease has a different immunological signature than doeschronic Crohn's disease. In aspects in which a patient presents withearly lesions, the microbial composition may be selected, with orwithout one or more prebiotics, to counteract a T helper cell 2-mediatedresponse. For example, the microbial composition, optionally combinedwith immunomodulatory molecules such as nucleotides or carbohydrates,may decrease interleukin-4 (IL-4) levels or increase IFN-γ. In aspectsin which a patient presents with chronic lesions, the microbialcomposition may be selected to counteract a T helper cell 1-mediatedresponse. For example, the microbial composition, optionally combinedwith immunomodulatory molecules such as nucleotides or carbohydrates,may decrease IL-2, IFN-γ, TNF-α, TL1A, IL-12, and/or IL-18. In someembodiments, in which a patient suffers from an IBD including but notlimited to Crohn's disease, a probiotic microbial composition, with orwithout one or more prebiotics, is administered to the patient such thatit is effective to reduce TNF-α levels, as detectable in feces samples,by approximately 5-fold, 10-fold, 25-fold, 50-fold, or 100-fold. Crohn'sdisease patients tend to present with low plasma levels of vitamins orminerals including but not limited to vitamin A, vitamin E, vitamin C,lycopene, carotenoids, and/or selenium. Patients eligible forimmunomodulatory treatment may thus be administered an immunomodulatorymicrobe, molecule, and/or microbial component optionally combined withan appropriate vitamin or mineral supplement, as determined by plasmadeficiency.

Probiotic compositions useful for treating or preventing Crohn's diseaseinclude, in exemplary embodiments, one or more bacterial strains fromTable 1. In other embodiments, the probiotic composition includes one ormore bacterial strains from Table 1A. In other embodiments, theprobiotic composition includes one or more bacterial strains from Table1B. In other embodiments, the probiotic composition includes one or morebacterial strains from Table 1C. In other embodiments, the probioticcomposition includes one or more bacterial strains from Table 1D. Inother embodiments, the probiotic composition includes one or morebacterial strains from Table 1E. In other embodiments, the probioticcomposition includes one or more bacterial strains from Table 1F. Insome embodiments, the probiotic composition contains a single strain ofbacteria. In other embodiments, the probiotic composition contains twoor more strains of bacteria, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more strains of bacteria.In other embodiments, the probiotic composition contains or isadministered in conjunction with a prebiotic, as described herein.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter.

Preferred bacterial genera also include Acetonema, Alkaliphilus,Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor,Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella,Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Halobacteroides, Heliobacterium, Heliophilum, Heliorestis,Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.),Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora,Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum,Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus,Thermoanaerobacter, and Thermosinus.

As provided herein, therapeutic compositions comprise, or in thealternative, modulate, the colonization and/or engraftment, of thefollowing exemplary bacterial entities: Lactobacillus gasseri,Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis,Enterococcus durans, Enterococcus villorum, Lactobacillus plantarum,Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcuscohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcusmitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcusanginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridiumsaccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6,Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcusgnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautiaproducta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonellaparvula, Clostridium methylpentosum, Clostridium islandicum,Faecalibacterium prausnitzii, Bacteroides uniformmis, Bacteroidesthetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus,Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirumpropionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria,Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta.

Preferred bacterial species are provided in Table 1, Table 1A, Table 1B,Table 1C, Table 1D, Table 1E, Table 1F, and Table 5. Optionally, in someembodiments, preferred bacterial species are spore formers. Wherespecific strains of a species are provided, one of skill in the art willrecognize that other strains of the species can be substituted for thenamed strain.

In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Acidaminococcusintestine. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isAcinetobacter baumannii. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Acinetobacter lwoffii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Akkermansia muciniphila. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Alistipes putredinis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Alistipes shahii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerostipes hadrus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerotruncus colihominis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides caccae.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidescellulosilyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isBacteroides dorei. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides eggerthii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides finegoldii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides fragilis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroides massiliensis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides ovatus.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidessalanitronis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBacteroides salyersiae. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides sp. 1_1_6. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides sp. 3_1_23. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides sp. D20. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroidesthetaiotaomicrond. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides uniformis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides vulgatus. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bifidobacterium adolescentis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacterium bifidum. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Bifidobacterium breve.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bifidobacteriumfaecale. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBifidobacterium kashiwanohense. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Bifidobacterium longum subsp. Longum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bifidobacterium stercoris. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Blautia (Ruminococcus) coccoides. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia faecis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia glucerasea. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)hansenii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)luti. In one embodiment, the bacterial entity, e.g., species or strain,useful in the compositions and methods of the invention is Blautia(Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia producta (Ruminococcus productus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)schinkii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia stercoris. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1).In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Blautia unculturedbacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia wexlerae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Candidatus Arthromitus sp.SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isCatenibacterium mitsuokai. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridiales bacterium1_7_47FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium asparagiforme. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridium bolteae. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium clostridioforme. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium glycyrrhizinilyticum. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium (Hungatella)hathewayi. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium histolyticum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium indolis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium leptum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium (Tyzzerella) nexile. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium perfringens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium(Erysipelatoclostridium) ramosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium scindens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium septum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium sp. 14774. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium sp.7_3_54FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium sp. HGF2. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium symbiosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Collinsella aerofaciens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Collinsella intestinalis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Coprobacillus sp. D7. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus catus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus comes. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Dorea formicigenerans.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Dorea longicatena.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Enterococcusfaecalis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEnterococcus faecium. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli S88. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacterium eligens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacteriumfissicatena. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEubacterium ramulus. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Eubacterium rectale. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Faecalibacterium prausnitzii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Flavonifractor plautii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Fusobacterium mortiferum.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Fusobacteriumnucleatum. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isHoldemania filiformis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Hydrogenoanaerobacterium saccharovorans. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Klebsiella oxytoca. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isLachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Lachnospiraceae bacterium 5_1_57FAA. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus casei. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus rhamnosus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Lactobacillus ruminis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Lactococcus casei.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Odoribactersplanchnicus. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isOscillibacter valericigenes. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Parabacteroides gordonii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Parabacteroides johnsonii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Parabacteroides merdae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Pediococcus acidilactici.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Peptostreptococcusasaccharolyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isPropionibacterium granulosum. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Roseburia intestinalis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Roseburia inulinivorans. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Ruminococcus faecis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus gnavus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus sp. ID8. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Ruminococcus torques.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Slackia piriformis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Staphylococcusepidermidis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStaphylococcus saprophyticus. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Streptococcus cristatus. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus dysgalactiae subsp. Equisimilis. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Streptococcus infantis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Streptococcusoralis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStreptococcus sanguinis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Streptococcus viridans. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus thermophiles. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Veillonella dispar.

In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Acidaminococcus intestine. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter baumannii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter lwoffii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Akkermansia muciniphila. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes putredinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes shahii. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Anaerostipes hadrus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Anaerotruncus colihominis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Bacteroides caccae. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides cellulosilyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides dorei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides eggerthii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides finegoldii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides fragilis. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Bacteroidesmassiliensis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Bacteroides ovatus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bacteroides salanitronis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides salyersiae. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 1_1_6. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 3_1_23. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. D20. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides thetaiotaomicrond. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides uniformis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides vulgatus. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium adolescentis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium bifidum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium breve. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium faecale. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium kashiwanohense. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium longum subsp. Longum.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Bifidobacteriumstercoris. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) coccoides. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia faecis. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiaglucerasea. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) hansenii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) luti. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) obeum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia producta (Ruminococcusproductus). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) schinkii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia stercoris. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiauncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia uncultured bacterium cloneSJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia wexlerae. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Catenibacterium mitsuokai. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridiaceae bacterium (Dielmafastidiosa) JC13. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Clostridialesbacterium 1_7_47FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumasparagiforme. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium bolteae.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium clostridioforme. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium glycyrrhizinilyticum. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Hungatella) hathewayi.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium histolyticum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium indolis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium leptum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Tyzzerella) nexile. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium perfringens. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Erysipelatoclostridium)ramosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumscindens. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium septum. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumsp. 14774. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.7_3_54FAA. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.HGF2. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumsymbiosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaaerofaciens. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaintestinalis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprobacillus sp.D7. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprococcus catus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Coprococcus comes. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea formicigenerans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea longicatena. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecalis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecium. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Erysipelotrichaceae bacterium 3_1_53. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Escherichia coli. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Escherichia coli S88. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium eligens. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium fissicatena. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium ramulus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium rectale. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Faecalibacterium prausnitzii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Flavonifractor plautii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium mortiferum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium nucleatum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Holdemania filiformis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Hydrogenoanaerobacterium saccharovorans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Klebsiella oxytoca. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Lachnospiraceaebacterium 7_1_58FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprisesLachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus casei. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus rhamnosus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus ruminis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactococcus casei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOdoribacter splanchnicus. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOscillibacter valericigenes. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides gordonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides johnsonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides merdae. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPediococcus acidilactici. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPeptostreptococcus asaccharolyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Propionibacterium granulosum. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia intestinalis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia inulinivorans. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus faecis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus gnavus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus sp. ID8. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus torques. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Slackia piriformis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus epidermidis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus saprophyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus cristatus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus dysgalactiae subsp. Equisimilis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus infantis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus oralis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus sanguinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus viridans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus thermophiles. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Veillonella dispar.

XI. Transplant Disorders and Graft Versus Host Disease (GVHD)

Graft-versus-host disease (GVHD) is a common complication for patientswho have received an allogeneic transplant (e.g., allogeneic bone marrowtransplant or allogeneic stem cell transplant). GVHD may also develop inpatients who have received a blood transfusion with blood products thathad not been irradiated. Patients suffering from GVHD have elevatedlevels of pro-inflammatory cytokines, particularly interleukin-6 (IL-6),as well as others including tumor necrosis factor-alpha (TNF-α),interleukin-1 (IL-1), interleukin-12 (IL-12), interferon-gamma (IFN-γ),and interleukin-2 (IL-2). GVHD is also associated with lowinterleukin-22 (IL-22) and the loss of interleukin-23 (IL-23)-responsiveor Lgr5+ innate lymphoid cells. Furthermore, GVHD patients have reducednumbers of intestinal stem cells, fewer or less active Paneth cells, andlower Foxp3 expression in T cells.

For over 40 years, it has been known that the bacteria inhabiting ourintestines are important modulators of the biology of hematopoietic celltransplantation (HCT) and impact the development of graft versus hostdisease. Studies in mice have shown reduction of GVHD withgut-decontaminating antibiotics (van Bekkum et al., 1974) andtransplantation in germ-free conditions (Jones et al., 1971). This ledto clinical attempts to eliminate intestinal bacterial colonization inallogenic BMT patients with a combination of gut decontaminationpractices and maintenance of a near-sterile environment (Storb et al.,1983). Initial clinical studies employing strategies to suppress theintestinal microbiota showed considerable promise, but later reportsfailed to demonstrate a clear and consistent benefit.

The recent understanding of the microbiota that inhabit the humangastrointestinal tract, skin, lungs, vagina, and other niches is beingappreciated in their role in health and disease of dysbiosis (e.g. seeHuman Microbiome Project Consortium 2012, Structure, function, anddiversity of the healthy human microbiome. Nature 486(7402):207-14).Several groups have reported preliminary data on the significance of thegut microbiota in HCT patients and in the development of GVHD.Collectively these reports show, using murine and human studies, thatthere is a significant decreased in diversity of gut microbiomefollowing HCT accompanied by marked changes in the population (Taur etal., 2012; Jenq et al., 2012; Taur et al., 2014). Most notably, we havefound that as much as 90% of allo BMT patients demonstrate a loss ofdiversity and a corresponding expansion of a single type of bacteriathat takes up much of the “space” within the intestinal floracompartment (Taur Y, et al. Clin Infect Dis. 2012).

Aspects of the invention are based, in part, on the realization thatmicrobes play an important role in the prevention, initiation anddevelopment of graft versus host disease (GVHD). The presence of certainmicrobial populations correlate with the initiation and development ofthe disease. More importantly, the presence of some species correlateswith reduced severity or mortality associated with the disease orprotection from GVHD altogether.

In some aspects, the invention is a composition or method for thetreatment of GVHD in patients suffering from chronic or acute GVHD. Incertain embodiments, the pharmaceutical composition, dosage form, or kitcomprises one or more microbes, with or without one or more prebiotics,capable of decreasing the expression or release of one or more of IL-6,IL-1, IL-12, IL-2, IFN-γ, and TNF-α. In some embodiments, thepharmaceutical composition, dosage form, or kit comprises one or moremicrobes capable of increasing the expression or release of IL-22. Themicrobial composition, with or without one or more prebiotics, may alsobe selected such that it is effective to stimulate or favor the survivalof Paneth cells, intestinal stem cells, FoxP3+ Treg cells (e.g., CD4+CD25+ FoxP3+ Treg cells), IL-23-responsive innate lymphoid cells, and/orLgr5+ innate lymphoid cells. In some embodiments, the microbialcomposition, with or without one or more prebiotics, regulatesexpression of Foxp3 by modulating (de)methylation of the Foxp3 locus. Inpreferred embodiments, the immunological tolerance elicited by themicrobial composition, with or without one or more prebiotics, does notreduce graft-vs-leukemia activity by the immune system.

In some aspects, the invention is a composition or method for theprevention of GVHD. Recent work has shown that small doses of IL-2 maybe able to restore T cell homeostasis in patients at risk for GVHD(Kennedy-Nasser et al., 2014. Ultra low-dose IL-2 for GVHD prophylaxisafter allogeneic hematopoietic stem cell transplantation mediatesexpansion of regulatory T cells without diminishing antiviral orantileukemic activity. Clin Cancer Res. 20:2215-2225). Thus in certainembodiments, the pharmaceutical composition, dosage form, or kitcomprises one or more microbes capable of slightly increasing IL-2levels in a patient such that the abundance of CD4+ CD25+ FoxP3+ Tregcells increases by 1.5-fold, 2-fold, or more than 5-fold. In a preferredembodiment, the IL-2-level enhancing treatment is administrated betweenone week and one month following the patient's transplant procedure.

Aspects of this invention are based at least in part on the discoverythat presence of certain bacterial populations can protect from GVHD andthat supplementation with such or similar bacterial compositions canprevent, treat or inhibit GVHD. Similarly, aspects of this inventionalso includes approaches used for augmentation of such bacterialcompositions using various techniques including substrates for bacterialfermentation and propagation as well as delivery of key end products ormetabolites of such bacteria which reconstitute needed functionality ofa bacterial community can also be used to prevent, treat or inhibitGVHD. Other aspects of this invention include agents for targetingbacteria that increase GVHD mortality or exacerbate clinical GVHD.

Herein, we disclose methods for identifying bacterial subsets thatincreased or decreased GVHD related mortality in order to identifysubsets that may modulate GVHD severity Stool specimen was collectedfrom patients who underwent conventional (non-T cell depleted) allo BMT.Fecal samples were collected and stored weekly over the course of thetransplant hospitalization this included prior to conditioning, as wellas on days 0, 7, 14, 21, 30, 60, and 100. GVHD was diagnosed clinically,confirmed pathologically by biopsy whenever possible, and classifiedaccording to standard criteria. Patients who engrafted were evaluablefor acute GVHD based on historical consensus criteria as describedpreviously (see Rowlings P A, Przepiorka D, Klein J P, et al. IBMTRSeverity Index for grading acute graft-versus-host disease:retrospective comparison with Glucksberg grade. Br J Haematol. 1997).These criteria were applied to GVHD with purely acute features thatoccurred after day 100. Cases of GVHD were further categorized bytreatment with or without systemic steroids (prednisone ormethylprednisolone, 0.5 mg/kg daily or higher). Cause of death wasdetermined using a standard algorithm where outcomes were prioritized inthe following order: 1) primary disease recurrence, 2) graft failure, 3)GVHD, 4) infection, and 5) organ failure; thus in patients withoutdisease recurrence or graft failure, those who were being treated forGVHD at the time of death were considered to have succumbed toGVHD-related mortality, including those who died from infections. Insome embodiments, the abundances of bacterial genera from patients whodid or did not die from GVHD by linear discriminant analysis (LDA)effect size (LEfSe) was compared to identify bacterial subsetsassociated with either increased or decreased GVHD-related mortality.

Provided herein are probiotic compositions of bacteria that modulateGVHD severity and related mortality and thus can be used to prevent,treat or inhibit GVHD. In one embodiment, a probiotic composition isadministered to a subject in an amount effective to increase short chainfatty acid production by one or more organisms in the gut of a mammalianhost.

In certain aspects, the invention relates to microbial compositions. Incertain embodiments, the microbial compositions can be used for theprevention, treatment or inhibition of GVHD. Aspects of the inventionrelate to microbial compositions that are being isolated from asubject's microbiota. In some embodiments, the subject is a healthymammal. In other embodiments, the subject is the recipient of thetransplant prior itself prior to inception of the conditioning regimen.In some embodiments, the microbial compositions comprise of bacteriathat are enriched in alive GVHD patients. These bacteria are stronglypredictive of improved overall survival following allo BMT and largelydriven by reduced GVHD-related mortality.

In some embodiments, the microbial composition comprises bacteria thatare associated with and can reduce clinical acute GVHD. In someembodiments, the microbial composition comprises of bacteria that canreduce acute GVHD grades 2-4. In some embodiments, the microbialcomposition comprises of bacteria that can reduce acute GVHD responsiveto treatment with systemic corticosteroids. In some embodiments, themicrobial composition comprises of bacteria that can reduce systemiccorticosteroid treatment refractory acute GVHD. In some embodiments, themicrobial composition comprises of bacteria that are associated withreduced lower gut GVHD. In some embodiments, the microbial compositioncomprises of bacteria that are associated with reduced liver GVHD. Insome embodiments, the microbial composition comprises of bacteria thatare associated with reduced skin GVHD.

In some embodiments, the microbial composition comprises bacteria thatare associated with and can reduce clinical chronic GVHD in a subject.In some embodiments, the subject has chronic GVHD. The subject withchronic GVHD may be receiving an immunosuppressive treatment. Theimmunosuppressive treatment may be one or more of methotrexate,cyclosporine, a corticosteroid, and antithymocyte globulin. Thecorticosteroid may be methylprednisolone. In some embodiments, thesubject requires or has required immunosuppressive treatment for aperiod of one or more years. In further embodiments, the subject hassteroid-refractory GVHD. The subject may be receiving one or more ofextracorporeal photophoresis, anti-TNF alpha antibody, mammalian targetof rapamycin (mTOR) inhibitor, mycophenolate mofetil, interleukin-2receptor antibody, alemtuzumab pentostatin, mesenchymal stem cells, andmethotrexate.

Aspects of this intervention also includes identification ofantimicrobial agents that increase GVHD incidence and severity byimpacting bacteria that are protective against GVHD. Identification ofsuch bacteria can be used as a guide to alter clinical practice toreduce GVHD incidence/severity. Antibiotics are used in BMT patientseither for gut decontamination purposes or to treat neutropenic fever.In some embodiments, BMT patients were analyzed to identify ifantibiotics they were administered such as piperacillin-tazobactam,imipenem-cilastatin, metronidazole, aztreonam and oral vancomycin leadto reduction in bacteria protective against GVHD including clostridialessuch as blautia. In some embodiments, BMT patients were analyzed toidentify if antibiotics they were administered such aspiperacillin-tazobactam, imipenem-cilastatin, metronidazole, aztreonamand oral vancomycin lead to increase in bacteria that exacerbate GVHD.In some embodiments, BMT patients were retroactively analyzed to see ifexposure to antibiotics with or without anaerobic coverage could impacton GVHD. In some embodiments, the microbial composition comprisesbacteria that are associated with GVHD-related mortality. Aspects ofthis invention also includes agents that targets bacterial populationsexacerbate clinical GVHD, increase incidence or severity of GVHD orincrease GVHD related mortality.

Aspects of this invention also includes approaches used for augmentationof bacterial compositions that prevent or mitigate GVHD using varioustechniques including substrates for bacterial fermentation andpropagation. It was noted that patients undergoing BMT can lose GVHDprotective bacteria without any exposure to antibiotics. This phenomenonwas also noted in murine models with experimental GVHD. In someembodiments, the composition of the invention comprises substrates thataugment bacterial compositions that prevent or mitigate GVHD. Given dietand nutrition has a tremendous impact on gut microbiome composition andthat oral nutrition intake is commonly reduced in allo BMT patients, theimpact of nutrition on GVHD protective bacteria abundance was analyzed.In some embodiments, the effect of reduced oral caloric consumption,particularly below 500 kcal/day, on Blautia abundance was analyzed. Apilot experiment of daily nutritional and flora monitoring in fivepatients undergoing allo BMT showed that a reduction in oral caloricconsumption, particularly below 500 kcal/day, was associated with areduction in Blautia abundance. Given blautia's ability to mitigateGVHD, a nutrition method to augment blautia levels in BMT patients wouldbe a rationale strategy for mitigating GVHD. The ability of blautia toferment a variety of sugars was thus analyzed using pH and opticaldensity to evaluate bacterial growth in media lacking glucose. In oneembodiment, the growth of blautia was analyzed. In another embodiment,bacteria that are potentially competing with blautia such aslactobacillus johnsonii. Lactobacillus johnsonii was evaluated.Lactobacillus johnsonii expands in the setting of calorie restriction atthe expense of Clostridia (Jenq et al., 2012) and is thus presumably adirect competitor for nutrients in the murine intestine. Using suchstrategies, specific substrates or sugars were identified that arefermentable by blautia but not lactobacillus. In one embodiment, thesubstrate that specifically augments blautia but not lactobacillus wasxylose. In another embodiment, the substrate that specifically augmentsblautia but not lactobacillus was rhamnose. In another embodiment, theeffect of administration of such substrates on blautia level wasinvestigated in experimental GVHD models. Administration of xylose inthe drinking water of mice was found to lead to an expansion of Blautiain the intestinal flora de-spite the presence of GVHD on day 14 afterBMT. In another embodiment, long term effect of xylose administrationwas evaluated to investigate effects on GVHD related survival. Long-termadministration of xylose led to improved survival of mice with GVHD.

Aspects of this invention also include compositions of bacterial endproducts or metabolites that are responsible or can impart thefunctionality of a bacterial compositions that prevent or mitigate GVHD.Short-chain fatty acids (SCFA), which are produced by many bacteria as abyproduct of carbohydrate fermentation. SCFA are one of the mostabundant metabolites produced by the gut microbiome, particularly theclass clostridia. SCFA have been found to be important modulators of theimmune system. In germ-free mice and vancomycin-treated conventionalmice, administration of SCFA (acetate, propionate, or butyrate) restorednormal numbers of Tregs in the large intestine (Smith P M, et al.Science. 2013; 569-573). In some embodiments, the SCFA levels of stoolspecimens from GVHD patients were analyzed for associations with blautiaabundance. Samples with reduced abundance of Blautia were also found tohave reduced abundance of the SCFA butyrate and acetate. In someembodiments, SCFA will be administered post BMT to reduce incidence andseverity of GVHD. In some embodiment, the SFCA administered is acetate.In some embodiment, the SFCA administered is butyrate while in otherembodiments it is propionate. In some embodiments, SCFA will reduce GVHDwithout impacting graft versus tumor effects. In some embodiments, SCFAadministration increases the number of peripheral tregs and leads toinduction of Foxp3 expression. In some embodiments, SCFA administrationreduces donor alloreactive T cells.

In some embodiments, metabolite profiles of patient tissue samples ormicrobes cultures from patient tissue are used to identify risk factorsfor developing an autoimmune or inflammatory response, to diagnose anautoimmune or inflammatory disease, to evaluate the prognosis orseverity of said disease, to evaluate the success of a treatmentregimen, or any combination thereof. Exemplary metabolites for thepurposes of diagnosis, prognostic risk assessment, or treatmentassessment purposes include short chain fatty acids, bile acids, andlactate. In preferred embodiments, metabolite profiles are taken atdifferent time points during a patient's disease and treatment in orderto better evaluate the patient's disease state including recovery orrelapse events. Such monitoring is also important to lower the risk of apatient developing a new autoimmune condition following immunomodulatorytreatment. In some embodiments, metabolite profiles inform subsequenttreatment. For example, patients at risk for developing GVHD andpresenting low levels of butyrate may be administered a microbialcomposition comprising microbes that produce butyrate (e.g., Blautiaspecies) and excluding microbes capable of depleting butyrate (e.g.Methanobacterium species). Probiotic compositions that produce SCFA inthe gut of a subject are particularly useful for the treatment of GVHD,because they improve intestinal barrier integrity, which is associatedwith improvement in overall survival in patients receiving a transplant.

In some embodiments, the administration is preventative or prophylacticin that the subject has not yet developed a detectable GVHD. In someembodiments, the preventative/prophylactic microbial composition will beadministered prior after the completion of conditioning region but priorto the transplant. Typically, it takes 2-3 weeks for engraftment of thetransplant to be completed. In some embodiments, thepreventative/prophylactic microbial composition will be administeredonce prior to transplant and then again on day 17 after completion ofantibiotics prescribed to prevent or treat neutropenic fever or otherinfections.

The classical definition of Graft versus host disease (GVHD) is that itis an immunological disorder in which the immune cells of a transplantattack the tissues of a transplant recipient and lead to organdysfunction. In the case of allogeneic bone marrow (BM) transplantation,T-cells from the transplanted BM recognize the host (the bonemarrow-transplanted patient i.e., the recipient) as non-self and attackits tissues and organs. The organs most commonly attacked are thegastrointestinal (GI) tract, skin, liver, and lungs. Historical data,however, says it is not only immune cells that are involved in diseasepathogenesis and points to the importance of the resident host gutmicrobes in the development of GVHD.

GVHD can be mild, moderate, or severe, depending on the extent of damageinflicted to different organs. The disease is divided into acute andchronic GVHD according to clinical manifestations. Patients with acuteGVHD typically suffer damage to the skin, GI tract, and liver. Skindamage ranges from redness to exfoliation. Insult to the GI tract canresult in bloody diarrhea and blood loss. Liver manifestations, thoughusually cholestatic in nature, can include liver failure in rare cases.

Acute GVHD usually develops within the first 100 days aftertransplantation, but it can also occur later. The clinicalmanifestations of chronic GVHD include red and itchy skin, dry eyes, drymouth, abnormal liver function with jaundice, and lung damage due tobronchiolitis obliterans. Chronic GVHD is the major cause of non-relapsemortality after allogeneic hematopoietic transplantations. Chronic GVHDusually develops more than 100 days after transplantation, but it canappear sooner.

Patients with chronic GVHD require prolonged immunosuppressivetreatment, averaging two to three years in length. The mechanismsunderlying chronic GVHD are considered to be somewhat distinct fromthose of acute GVHD. Thus, chronic GVHD is not simply an end-stage ofacute GVHD.

Clinical Staging of Acute GVHD (aGVHD)—There are two systems forquantifying the severity of aGVHD, namely, the International Bone MarrowTransplant Registry (IBMTR) grading system and the Glucksberg gradingsystem. For both systems, the stage of aGVHD is first determinedseparately in the three main target organs (skin, liver and gut). Thesegrades are then used to determine an overall aGVHD grade, using eitherthe International Bone Marrow Transplant Registry (IBMTR) or Glucksbergcriteria.

For each grading system, the acute GVHD stage for each target organ isfirst determined according to certain clinical measures, as provided inTable 3.

The invention also provides, in one aspect, a method of increasing theduration of survival of a subject receiving a bone marrow transplant, byadministering to the subject a probiotic composition comprising anisolated bacterial population, such that the duration of survival of thesubject is increased. In a preferred embodiment, the bacterialpopulation is a human-derived bacterial population. A human-derivedbacterial population includes bacterial strains that natively inhabit ahuman host, as opposed to a non-human mammalian host. Administration ofthe probiotic composition can reduce the likelihood that the subjectwill develop sepsis following the bone marrow transplant. Administrationof the probiotic composition can also reduce the likelihood that thesubject will develop graft versus host disease (GVHD) following the bonemarrow transplant. The probiotic composition can be administered to thesubject prior to, after, or concurrently with receiving the bone marrowtransplant.

In one embodiment, the probiotic composition reduces intestinalpermeability in the subject. This can be achieved by, for example,administering a probiotic composition that contains bacteria whichproduce short chain fatty acids, which increase intestinal barrierintegrity in a subject. In exemplary embodiments, the bacteria producebutyrate, acetate, propionate, or valerate, or combinations thereof.Also provided is a method of reducing intestinal permeability in asubject receiving a transplant, comprising administering to the subjecta probiotic composition comprising an isolated bacterial population anda pharmaceutically acceptable excipient, such that the intestinalpermeability of the subject of the subject receiving the transplant isreduced.

In another embodiment, the probiotic composition reduces inflammation inthe subject, e.g., in the gastrointestinal tract, or in a distallocation. The probiotic composition can contain anti-inflammatorybacteria. Anti-inflammatory bacteria are described herein. For example,anti-inflammatory bacteria included in the probiotic composition candecrease secretion of pro-inflammatory cytokines (e.g., IFNγ, IL-12p70,IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα, and combinations thereof)and/or increase secretion of anti-inflammatory cytokines (e.g., IL-10,IL-13, IL-4, IL-5, TGFβ, and combinations thereof) by human host cells,such as human epithelial cells or immune cells, e.g., peripheral bloodmononuclear cells (PBMCs). Bacteria which produce short chain fattyacids also have anti-inflammatory properties.

In some embodiments, the subject has received or will receive ahematopoietic stem cell transplant. In some embodiments, the subjectwill receive a hematopoietic stem cell transplant that is T celldepleted.

In exemplary embodiments, the subject has a disorder such as ahematopoietic neoplastic disorder, leukemia, lymphoma, and multiplemyeloma. In some embodiments, the subject has a hematopoietic neoplasticdisorder. The subject may have leukemia. The leukemia may be chronicmyelogeneous leukemia or chronic lymphocytic leukemia. In someembodiments, the subject has lymphoma. The lymphoma may be Hodgkin'sdisease or non-Hodgkin's lymphoma. In some cases, the subject hasmultiple myeloma. The subject may receive or will receive a bone marrow,peripheral blood stem cell, or cord blood transplant. In someembodiments, the subject has received or will receive whole bodyirradiation. In further embodiments, the subject is female.

Due to the unique mechanism of action of probiotic compositions in thetreatment or prevention of GVHD, probiotic compositions can be selectedwhich minimize GVHD, but do not significantly reduce or eliminate thegraft versus tumor (GVT) effect of the bone marrow transplant.

In other embodiments, the subject receiving the transplant has anautoimmune disorder, such as, for example, lupus, multiple sclerosis,systemic sclerosis, Crohn's disease, type I diabetes, and juvenileidiopathic arthritis. In another embodiment, the subject receiving thetransplant has sickle cell disease or sickle cell anemia.

In an exemplary embodiment, the invention provides a method ofincreasing the duration of survival of a subject receiving a bone marrowtransplant, comprising administering to the subject a probioticcomposition comprising an isolated population of anti-inflammatorybacteria capable of decreasing secretion of pro-inflammatory cytokinesand/or increasing secretion of anti-inflammatory cytokines by humanperipheral blood mononuclear cells (PBMCs), and a pharmaceuticallyacceptable excipient, in an amount effective to reduce inflammation inthe gastrointestinal tract of the subject, such that the duration ofsurvival of the subject is increased.

In some embodiments, the subject has received or will receive atransplant from an HLA-matched related donor or an HLA-matched unrelateddonor. The subject may also have received or may receive a transplantfrom an HLA-mismatched related or unrelated donor. In some embodiments,the subject will receive an autologous transplant. In some embodiments,the microbial composition will augment an autologous or allogeneictransplant. In some embodiments, the microbial composition will improveengraftment after an autologous or allogeneic transplant. In someembodiments, the microbial composition will improve neutropenic recoveryafter an autologous or allogeneic transplant. In some embodiments, themicrobial composition will reduce complications after an allogeneictransplant. In some embodiments, the microbial composition will reducecomplications after an autologous transplant. Complications afterallogeneic transplant may include but are not limited to infections,organ failure, Veno-occlusive disease (VOD) of the liver, and/orInterstitial Pneumonia Syndrome (IPS).

In some embodiments, the subject will receive GVHD prophylaxis regimenthat are standardly used in the clinic in addition to the microbialcomposition. This may include administering immunosuppressive treatmentsuch as methotrexate, cyclosporine, corticosteroids, or anti-thymocyteglobulin.

In some embodiments, the subject will receive GVHD treatment regimenthat are standardly used in the clinic in addition to the microbialcomposition for management of graft versus host disease. The jointworking group established by the Haemato-oncology subgroup of theBritish Committee for Standards in Haematology (BCSH) and the BritishSociety for Bone Marrow Transplantation (BSBMT) reviewed the availableliterature and made recommendations in 2012 for the management of acutegraft-versus-host disease. Their recommendations are as follows: (1) Themanagement of grade I disease should include topical therapy andoptimizing levels of calcineurin inhibitors without the need foradditional systemic immunosuppression. (2) The use of systemiccorticosteroids is recommended for first line therapy for grade II-IVGVHD. (3) The following agents are suggested for use in the second linetreatment of steroid-refractory acute GVHD: extracorporealphotopheresis, anti-tumour necrosis factor a antibodies, mammaliantarget of rapamycin (mTOR) inhibitors, mycophenolate mofetil,interleukin-2 receptor antibodies. (4) The following agents aresuggested as third line treatment options in acute steroid-refractoryGVHD: alemtuzumab pentostatin, mesenchymal stem cells and methotrexate.In one aspect, disclosed herein are methods of treating, inhibiting, orpreventing GVHD in a subject wherein the subject has acutesteroid-refractory GVHD. In such methods, the method can furthercomprise administering to the subject extracorporeal photopheresis,anti-tumour necrosis factor a antibodies, mammalian target of rapamycin(mTOR) inhibitors, mycophenolate mofetil, interleukin-2 receptorantibodies, alemtuzumab pentostatin, mesenchymal stem cells,methotrexate, or any combination thereof.

In other embodiments, the subject is administered a prebioticcomposition in conjunction with the probiotic composition. For example,in one aspect, the invention provides method of increasing the durationof survival of a subject receiving a bone marrow transplant, byadministering to the subject a probiotic composition comprising anisolated bacterial population, wherein the probiotic composition reducesintestinal permeability in the subject, and administering a prebioticthat enhances the activity of the bacterial population, such that theduration of survival of the subject is increased. Prebiotic compositionsof the invention are described herein. Exemplary prebiotics are providedin Table 7, and in FIG. 29.

The invention also provides, in certain aspects, method of preventing ortreating graft versus host disease (GVHD) in a subject receiving atransplant, comprising administering to the subject a probioticcomposition comprising an isolated bacterial population, such that GVHDis prevented or treated. The probiotic composition can increase theduration of survival of the transplant recipient, by preventing GVHD,and/or preventing the development of sepsis. Preferably the bacterialpopulation is a human-derived bacterial population. As noted above, thesubject may be receiving a hematopoietic stem cell transplant or a bonemarrow transplant. In other embodiments, the subject is receiving asolid organ transplant.

GVHD commonly develops after an allogeneic bone marrow transplant (BMT)but it can also appear after solid organ transplantation. The exactincidence rate of GVHD after solid organ transplantation is unknown.Mild cases likely remain undiagnosed because the clinical features offever, rash, and diarrhea can be misinterpreted as related topost-transplantation infections. The incidence rate of GVHD is highestafter small bowel transplantation (about 5%), followed by livertransplantation. But in general, the incidence rate for solid organtransplantation is very small relative to bone marrow transplantation.In embodiments, the microbial composition is used to prevent or treatGVHD in solid organ transplant recipients.

In some embodiments, the microbial composition will be administered tosubjects receiving solid organ transplantation. Transplanted solidorgans may include a kidney, heart, skin, a lung, a liver, a pancreas,an intestine, an endocrine gland, a bladder, or a skeletal muscle. Insome embodiments, microbial composition will be used to prevent graftrejection in a recipient of a transplanted solid organ. In someembodiments, microbial composition will be used to prevent othercomplications of solid organ transplantation such as infections.

In exemplary embodiments, the subject has a hematopoietic neoplasticdisorder such as, for example, leukemia, lymphoma, or multiple myeloma,an autoimmune disorder such as, for example, lupus, multiple sclerosis,systemic sclerosis, Crohn's disease, type I diabetes, or juvenileidiopathic arthritis, or a sickle cell disorder such as, for example,sickle cell disease or sickle cell anemia.

Aspects of this invention also include an immune mechanism via whichacute or chronic GVHD or solid organ transplant recipients are managed.In some embodiments, a test article inhibits the functionality ofantigen presenting cells such as dendritic cells where the test articleis the microbial composition, prebiotics, microbial composition plusprebiotics or microbial metabolites. In some embodiments, a test articleinhibits maturation of antigen presenting cells such that levels ofCD40, CD80, CD86, PD-L1 and PD-L2 are modulated. In some embodiments,test article inhibits activity of antigen presenting cells such thatproduction of cytokines such as TGFβ, IL-10, IL-4, IL-12 are modulated.In some embodiments, test article inhibits activity of antigenpresenting cells such as their endocytic/phagocytic capacity ishindered. In some embodiments, test article inhibits activity of antigenpresenting cells such that their ability to activate naïve T cells ishindered.

In some embodiments, test article inhibits the functionality of T cellswhere the test article is the microbial composition, prebiotics,microbial composition plus prebiotics or microbial metabolites. In someembodiments, test article alters the functionality of CD4+ T cells suchthat their activation status is altered affecting surface levels ofCD25. In some embodiments, test article alters the functionality of CD4+T cells such that their proliferative capacity is inhibited. In someembodiments, test article increases the number and differentiation ofperipheral regulatory T cells. In some embodiments, the test articleaffects production of cytokines by T cells such as but not limited toIL-6, TNF-alpha, IFN-γ, IL-10, IL-4. In some embodiments, the testarticle reduces the cytotoxic capacity of effector CD8+ cells.

As described herein, reducing systemic or local inflammation in asubject reduces the likelihood that a subject will develop GVHD.Accordingly, in another embodiment, the invention provides a method ofreducing inflammation in the gastrointestinal tract of a subjectreceiving a transplant, by administering to the subject a probioticcomposition comprising an isolated, anti-inflammatory bacterialpopulation and a pharmaceutically acceptable excipient, such thatinflammation in the gastrointestinal tract of the subject receiving thetransplant is reduced. Probiotic compositions containinganti-inflammatory bacterial populations described herein are suitablefor the practice of this embodiment.

Additional probiotic compositions useful for treatment or prevention ofGVHD contain bacterial strains capable of reducing inflammation in asubject. Such immunomodulatory (anti-inflammatory) bacteria can modulatecytokine expression by host immune cells, resulting in an overallincrease in secretion of anti-inflammatory cytokines and/or an overalldecrease in secretion of pro-inflammatory cytokines, systemicallyreducing inflammation in the subject. In exemplary embodiments,probiotic compositions useful for treatment or prevention of GVHDstimulate secretion of one or more anti-inflammatory cytokines by hostimmune cells, such as PBMCs. Anti-inflammatory cytokines include, butare not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGFβ, andcombinations thereof. In other exemplary embodiments, probioticcompositions useful for treatment or prevention of GVHD inhibitsecretion of one or more pro-inflammatory cytokines by host immunecells, such as PBMCs. Pro-inflammatory cytokines include, but are notlimited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα,and combinations thereof. Other exemplary cytokines are known in the artand are described herein. Probiotic compositions containinganti-inflammatory bacteria reduce inflammation and restore barrierfunction at the site of administration, e.g., in the gastrointestinaltract, as well as at distal sites throughout the body of the subject.

Other exemplary probiotic compositions useful for treatment orprevention of GVHD contain bacterial strains capable of altering theproportion of immune subpopulations, e.g., T cell subpopulations, in thesubject.

For example, immunomodulatory bacteria can increase or decrease theproportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in asubject. The increase or decrease in the proportion of immune cellsubpopulations may be systemic, or it may be localized to a site ofaction of the probiotic, e.g., in the gastrointestinal tract or at thesite of a distal dysbiosis. In some embodiments, a probiotic compositioncomprising immunomodulatory bacteria is used for treatment or preventionof GVHD based on the desired effect of the probiotic composition on thedifferentiation and/or expansion of subpopulations of immune cells inthe subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria that increase the proportion of Treg cells in a subject. Inanother embodiment, a probiotic composition contains immunomodulatorybacteria that decrease the proportion of Treg cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th17 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th17 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th1 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th1 cells in a subject. In oneembodiment, a probiotic composition contains immunomodulatory bacteriathat increase the proportion of Th2 cells in a subject. In anotherembodiment, a probiotic composition contains immunomodulatory bacteriathat decrease the proportion of Th2 cells in a subject.

In one embodiment, a probiotic composition contains immunomodulatorybacteria capable of modulating the proportion of one or more of Tregcells, Th17 cells, Th1 cells, and combinations thereof in a subject.Certain immune cell profiles may be particularly desirable to treat orprevent GVHD. For example, in some embodiments, treatment or preventionof GVHD can be promoted by increasing numbers of Treg cells and Th2cells, and decreasing numbers of Th17 cells and Th1 cells. Accordingly,probiotic compositions for the treatment or prevention of GVHD maycontain probiotics capable of promoting Treg cells and Th2 cells, andreducing Th17 and Th1 cells.

Probiotic compositions useful for treating or preventing GVHD include,in exemplary embodiments, one or more bacterial species from Table 1. Inother embodiments, the probiotic composition includes one or morebacterial species from Table 1A. In other embodiments, the probioticcomposition includes one or more bacterial species from Table 1B. Inother embodiments, the probiotic composition includes one or morebacterial species from Table 1C. In other embodiments, the probioticcomposition includes one or more bacterial species from Table 1D. Inother embodiments, the probiotic composition includes one or morebacterial species from Table 1E. In other embodiments, the probioticcomposition includes one or more bacterial species from Table 1F. Inother embodiments, the probiotic composition includes one or morebacterial species from Table 5. In some embodiments, the probioticcomposition contains a single species of bacteria. In other embodiments,the probiotic composition contains two or more species of bacteria,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 500, 1000 or more species of bacteria. In one embodiment, theprobiotic composition contains no more than 20 species of bacteria,e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 species of bacteria. In exemplary embodiments, the probioticcomposition contains 8 bacterial species. In other exemplaryembodiments, the probiotic composition contains 9 bacterial species. Inother embodiments, the probiotic composition contains or is administeredin conjunction with a prebiotic, as described herein.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, and Turicibacter.

Preferred bacterial genera also include Acetonema, Alkaliphilus,Amphibacillus, Ammonifex, Anaerobacter, Caldicellulosiruptor,Caloramator, Candidatus, Carboxydibrachium, Carboxydothermus, Cohnella,Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Halobacteroides, Heliobacterium, Heliophilum, Heliorestis,Lachnoanaerobaculum, Lysinibacillus, Oceanobacillus, Orenia (S.),Oxalophagus, Oxobacter, Pelospora, Pelotomaculum, Propionispora,Sporohalobacter, Sporomusa, Sporosarcina, Sporotomaculum,Symbiobacterium, Syntrophobotulus, Syntrophospora, Terribacillus,Thermoanaerobacter, and Thermosinus.

In one embodiment, a probiotic composition for the treatment orprevention of GVHD consists essentially of Blautia.

In another embodiment, a probiotic composition for the treatment orprevention of GVHD does not contain Blautia alone.

As provided herein, therapeutic compositions comprise, or in thealternative, modulate, the colonization and/or engraftment, of thefollowing exemplary bacterial entities: Lactobacillus gasseri,Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecalis,Enterococcus durans, Enterococcus villorum, Lactobacillus plantarum,Pediococcus acidilactici, Staphylococcus pasteuri, Staphylococcuscohnii, Streptococcus sanguinis, Streptococcus sinensis, Streptococcusmitis, Streptococcus sp. SCA22, Streptococcus sp. CR-3145, Streptococcusanginosus, Streptococcus mutans, Coprobacillus cateniformis, Clostridiumsaccharogumia, Eubacterium dolichum DSM 3991, Clostridium sp. PPf35E6,Clostridium sordelli ATCC 9714, Ruminococcus torques, Ruminococcusgnavus, Clostridium clostridioforme, Ruminococcus obeum, Blautiaproducta, Clostridium sp. ID5, Megasphaera micronuciformis, Veillonellaparvula, Clostridium methylpentosum, Clostridium islandicum,Faecalibacterium prausnitzii, Bacteroides uniformmis, Bacteroidesthetaiotaomicron, Bacteroides acidifaciens, Bacteroides ovatus,Bacteroides fragilis, Parabacteroides distasonis, Propinionibacteirumpropionicum, Actinomycs hyovaginalis, Rothia mucilaginosa, Rothia aeria,Bifidobacterium breve, Scardovia inopinata and Eggerthella lenta.

Preferred bacterial species are provided in Table 1, Table 1A, Table 1B,Table 1C, Table 1D, Table 1E, Table 1F, and Table 5. Optionally, in someembodiments, preferred bacterial species are spore formers. Wherespecific strains of a species are provided, one of skill in the art willrecognize that other strains of the species can be substituted for thenamed strain.

In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Acidaminococcusintestine. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isAcinetobacter baumannii. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Acinetobacter lwoffii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Akkermansia muciniphila. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Alistipes putredinis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Alistipes shahii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerostipes hadrus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Anaerotruncus colihominis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides caccae.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidescellulosilyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isBacteroides dorei. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides eggerthii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides finegoldii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides fragilis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroides massiliensis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroides ovatus.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bacteroidessalanitronis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBacteroides salyersiae. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides sp. 1_1_6. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides sp. 3_1_23. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bacteroides sp. D20. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bacteroidesthetaiotaomicrond. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bacteroides uniformis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Bacteroides vulgatus. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Bifidobacterium adolescentis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacterium bifidum. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Bifidobacterium breve.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Bifidobacteriumfaecale. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBifidobacterium kashiwanohense. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Bifidobacterium longum subsp. Longum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Bifidobacterium stercoris. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Blautia (Ruminococcus) coccoides. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia faecis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia glucerasea. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)hansenii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)luti. In one embodiment, the bacterial entity, e.g., species or strain,useful in the compositions and methods of the invention is Blautia(Ruminococcus) obeum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia producta (Ruminococcus productus). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia (Ruminococcus)schinkii. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia stercoris. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1). In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isBlautia uncultured bacterium clone SJTU_B_14_30 (GenBank: EF402926.1).In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Blautia unculturedbacterium clone SJTU_C_14_16 (GenBank: EF404657.1). In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Blautia wexlerae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Candidatus Arthromitus sp.SFB-mouse-Yit. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isCatenibacterium mitsuokai. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridiaceae bacterium (Dielma fastidiosa) JC13. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridiales bacterium1_7_47FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium asparagiforme. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Clostridium bolteae. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium clostridioforme. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium glycyrrhizinilyticum. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium (Hungatella)hathewayi. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium histolyticum. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium indolis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium leptum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium (Tyzzerella) nexile. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium perfringens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium(Erysipelatoclostridium) ramosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Clostridium scindens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Clostridium septum. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Clostridium sp. 14774. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Clostridium sp.7_3_54FAA. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium sp. HGF2. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Clostridium symbiosum. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Collinsella aerofaciens. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Collinsella intestinalis. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Coprobacillus sp. D7. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus catus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Coprococcus comes. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Dorea formicigenerans.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Dorea longicatena.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Enterococcusfaecalis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEnterococcus faecium. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Erysipelotrichaceae bacterium 3_1_53. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Escherichia coli S88. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacterium eligens. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Eubacteriumfissicatena. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isEubacterium ramulus. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Eubacterium rectale. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Faecalibacterium prausnitzii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Flavonifractor plautii. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Fusobacterium mortiferum.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Fusobacteriumnucleatum. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isHoldemania filiformis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Hydrogenoanaerobacterium saccharovorans. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Klebsiella oxytoca. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isLachnospiraceae bacterium 7_1_58FAA. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Lachnospiraceae bacterium 5_1_57FAA. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus casei. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Lactobacillus rhamnosus. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Lactobacillus ruminis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Lactococcus casei.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Odoribactersplanchnicus. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isOscillibacter valericigenes. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Parabacteroides gordonii. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Parabacteroides johnsonii. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Parabacteroides merdae. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Pediococcus acidilactici.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Peptostreptococcusasaccharolyticus. In one embodiment, the bacterial entity, e.g., speciesor strain, useful in the compositions and methods of the invention isPropionibacterium granulosum. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Roseburia intestinalis. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Roseburia inulinivorans. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Ruminococcus faecis. In one embodiment,the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus gnavus. In oneembodiment, the bacterial entity, e.g., species or strain, useful in thecompositions and methods of the invention is Ruminococcus sp. ID8. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Ruminococcus torques.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Slackia piriformis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Staphylococcusepidermidis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStaphylococcus saprophyticus. In one embodiment, the bacterial entity,e.g., species or strain, useful in the compositions and methods of theinvention is Streptococcus cristatus. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus dysgalactiae subsp. Equisimilis. Inone embodiment, the bacterial entity, e.g., species or strain, useful inthe compositions and methods of the invention is Streptococcus infantis.In one embodiment, the bacterial entity, e.g., species or strain, usefulin the compositions and methods of the invention is Streptococcusoralis. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isStreptococcus sanguinis. In one embodiment, the bacterial entity, e.g.,species or strain, useful in the compositions and methods of theinvention is Streptococcus viridans. In one embodiment, the bacterialentity, e.g., species or strain, useful in the compositions and methodsof the invention is Streptococcus thermophiles. In one embodiment, thebacterial entity, e.g., species or strain, useful in the compositionsand methods of the invention is Veillonella dispar.

In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Acidaminococcus intestine. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter baumannii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Acinetobacter lwoffii. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Akkermansia muciniphila. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes putredinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Alistipes shahii. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Anaerostipes hadrus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Anaerotruncus colihominis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Bacteroides caccae. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides cellulosilyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Bacteroides dorei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides eggerthii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides finegoldii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBacteroides fragilis. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Bacteroidesmassiliensis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Bacteroides ovatus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bacteroides salanitronis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides salyersiae. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 1_1_6. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. 3_1_23. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides sp. D20. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides thetaiotaomicrond. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides uniformis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bacteroides vulgatus. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium adolescentis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium bifidum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium breve. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium faecale. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium kashiwanohense. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Bifidobacterium longum subsp. Longum.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Bifidobacteriumpseudocatenulatum. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Bifidobacteriumstercoris. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) coccoides. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia faecis. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiaglucerasea. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) hansenii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) luti. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia (Ruminococcus) obeum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia producta (Ruminococcusproductus). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia(Ruminococcus) schinkii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesBlautia stercoris. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Blautiauncultured bacterium clone BKLE_a03_2 (GenBank: EU469501.1). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia uncultured bacterium cloneSJTU_B_14_30 (GenBank: EF402926.1). In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1). In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Blautia unculturedbacterium clone S1-5 (GenBank: GQ898099.1). In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2). In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Blautia wexlerae. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Candidatus Arthromitus sp. SFB-mouse-Yit. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Catenibacterium mitsuokai. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridiaceae bacterium (Dielmafastidiosa) JC13. In one embodiment, the bacterial population useful inthe compositions and methods of the invention comprises Clostridialesbacterium 1_7_47FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumasparagiforme. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium bolteae.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium clostridioforme. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium glycyrrhizinilyticum. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Hungatella) hathewayi.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Clostridium histolyticum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium indolis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium leptum. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Tyzzerella) nexile. Inone embodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium perfringens. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Clostridium (Erysipelatoclostridium)ramosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumscindens. In one embodiment, the bacterial entity, e.g., species orstrain, useful in the compositions and methods of the invention isClostridium septum. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprises Clostridiumsp. 14774. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.7_3_54FAA. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridium sp.HGF2. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Clostridiumsymbiosum. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaaerofaciens. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Collinsellaintestinalis. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprobacillus sp.D7. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Coprococcus catus.In one embodiment, the bacterial population useful in the compositionsand methods of the invention comprises Coprococcus comes. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea formicigenerans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Dorea longicatena. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecalis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Enterococcus faecium. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Erysipelotrichaceae bacterium 3_1_53. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Escherichia coli. In one embodiment,the bacterial population useful in the compositions and methods of theinvention comprises Escherichia coli S88. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium eligens. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium fissicatena. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium ramulus. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Eubacterium rectale. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Faecalibacterium prausnitzii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Flavonifractor plautii. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium mortiferum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Fusobacterium nucleatum. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Holdemania filiformis. In one embodiment, thebacterial population useful in the compositions and methods of theinvention comprises Hydrogenoanaerobacterium saccharovorans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Klebsiella oxytoca. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Lachnospiraceae bacterium3_1_57FAA_CT1. In one embodiment, the bacterial population useful in thecompositions and methods of the invention comprises Lachnospiraceaebacterium 7_1_58FAA. In one embodiment, the bacterial population usefulin the compositions and methods of the invention comprisesLachnospiraceae bacterium 5_1_57FAA. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus casei. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus rhamnosus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactobacillus ruminis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Lactococcus casei. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOdoribacter splanchnicus. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesOscillibacter valericigenes. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides gordonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides johnsonii. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesParabacteroides merdae. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPediococcus acidilactici. In one embodiment, the bacterial populationuseful in the compositions and methods of the invention comprisesPeptostreptococcus asaccharolyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Propionibacterium granulosum. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia intestinalis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Roseburia inulinivorans. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus faecis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus gnavus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus sp. ID8. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Ruminococcus torques. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Slackia piriformis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus epidermidis. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Staphylococcus saprophyticus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus cristatus. In one embodiment, the bacterialpopulation useful in the compositions and methods of the inventioncomprises Streptococcus dysgalactiae subsp. Equisimilis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus infantis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus oralis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus sanguinis. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus viridans. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Streptococcus thermophiles. In oneembodiment, the bacterial population useful in the compositions andmethods of the invention comprises Veillonella dispar.

XII. Diagnostic Methods

In some embodiments, metabolite profiles of patient tissue samples ormicrobes cultures from patient tissue are used to identify risk factorsfor developing an autoimmune or inflammatory response, to diagnose anautoimmune or inflammatory disease, to evaluate the prognosis orseverity of said disease, to evaluate the success of a treatmentregimen, or any combination thereof. Exemplary metabolites for thepurposes of diagnosis, prognostic risk assessment, or treatmentassessment purposes include short chain fatty acids, bile acids, andlactate. In preferred embodiments, metabolite profiles are taken atdifferent time points during a patient's disease and treatment in orderto better evaluate the patient's disease state including recovery orrelapse events. Such monitoring is also important to lower the risk of apatient developing a new autoimmune condition following immunomodulatorytreatment. In some embodiments, metabolite profiles inform subsequenttreatment. For example, patients at risk for developing GVHD andpresenting low levels of butyrate may be administered a microbialcomposition comprising microbes that produce butyrate (e.g., Blautiaspecies) and excluding microbes capable of depleting butyrate (e.g.Methanobacterium species). In another example, patients experiencingbacterial vaginosis—which increases the risk that a woman will sufferfrom a sexually transmitted disease or experience fertility issues—oftenpresents with abnormally low lactic acid levels. Thus, patients with lowlactic acid production in the vagina may be administered a microbialcomposition comprising lactic acid producing microbes (e.g.,Lactobacillus species) to restore a healthy microbiome state.

Patient Selection.

Particular bacterial compositions can be selected for individualpatients or for patients with particular profiles. For example, 16Ssequencing can be performed for a given patient to identify the bacteriapresent in his or her microbiota. The sequencing can either profile thepatient's entire microbiome using 16S sequencing (to the family, genera,or species level), a portion of the patient's microbiome using 16Ssequencing, or it can be used to detect the presence or absence ofspecific candidate bacteria that are biomarkers for health or aparticular disease state, such as markers of multi-drug resistantorganisms or specific genera of concern such as Escherichia. Based onthe biomarker data, a particular composition can be selected foradministration to a patient to supplement or complement a patient'smicrobiota in order to restore health or treat or prevent disease. Inanother embodiment, patients can be screened to determine thecomposition of their microbiota to determine the likelihood ofsuccessful treatment.

XIII. Kits

In certain aspects, the invention relates to kits for the treatment ofan autoimmune disease and/or inflammatory disease. The kits may comprisea microbial composition and an immunomodulatory carbohydrate, aprebiotic, microbial DNA, a mucolytic agent or a combination thereof.Optionally, the microbial composition, the immunomodulatorycarbohydrate, the prebiotic, microbial DNA, and/or the mucolytic agentare matched to exhibit a synergistic treatment effect in a subject whenemploying an appropriate treatment regimen or preventative measure foran autoimmune and/or inflammatory disease.

The kits provided may comprise one or more containers. The containersmay comprise singly isolated microbial compositions comprising one ormore microbes and/or singly isolated prebiotic compositions comprisingone or more carbohydrates. The microbial compositions, with or withoutone or more prebiotics, in the different containers may be administeredat the same time or at different times, and may be administered in aspecific order.

The compositions may, optionally, additively, or synergistically provideimmunomodulatory effects when administered to a subject. The microbialcomposition, with or without one or more prebiotics, may comprise livemicrobes, microbes that are lyophilized, freeze-dried, and/orsubstantially dehydrated, or the composition may comprise bacterial orfungal spores or virions. In some embodiments, the kit further comprisesan effective amount of one or more immunomodulary carbohydrates in oneor more containers. In some embodiments, the kit further comprises inone or more containers an effective amount of an anti-mucolytic agent.In some embodiments, the kit further comprises one or more containers aneffective amount of a prebiotic. In some embodiments, the kit furthercomprises an effective amount of a pro-inflammatory or anti-inflammatoryagent. In some embodiments, the kit further comprises a pharmaceuticallyacceptable excipient or diluent.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Theentire contents of all references, patents, and published patentapplications cited throughout this application are hereby incorporatedby reference in their entirety.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press, Vols A and B, 1992). Enzyme Linked Immunosorbent Assays(ELISAs) and Western blots described below are performed using kitsaccording to the manufacturers' (e.g., Life Technologies, Thermo FisherScientific, New York, USA) instructions.

Example 1. Assessment of Intestinal Permeability after Administration ofBacteria, Prebiotic or Combinations Thereof

The main function of the gastrointestinal (GI) tract is to digest andabsorb nutrients from food. The mucosa of the GI tract forms a selectivebarrier between the host and the environment of the gut lumen. Themucosa allows transport of nutrients while restricting passage of largermolecules and bacteria. Impaired barrier integrity is believed tocontribute to the pathogenesis of many disorders including autoimmunediseases, including transplant disorders such asgraft-versus-host-disease (GVHD), and neurological disorders. Disruptionof the intestinal barrier due to toxins, dysbiosis, inflammation orother factors is believed to result in the passage and presentation ofenvironmental antigens to the immune system leading to aberrant immuneresponses. Similarly, the leakage of bacterial endotoxin or other toxicmetabolites into the circulation can lead to systemic inflammationpromoting the development of autoimmunity and neuroinflammation.

Restoration of GI barrier integrity through the administration ofselected prebiotics and/or probiotics represents an approach to correcta basic defect underlying multiple pathological conditions.

In a first set of experiments, intestinal permeability was assessedusing serum endotoxin levels as a marker of gut permeability in micetreated with xylose and/or antibiotics. Basal levels of intestinalpermeability can be measured under disease or normal conditions.Intestinal permeability can be induced in mice through administration ofinflammatory stimuli such as cholera toxin (3 oral gavages of 10 μgcholera toxin, 5 days apart), Poly I:C (3 intraperioneal injections of 1mg/kg, 3 days apart) or dextran sulfate (3% dextran sulfate sodium saltin drinking water for 7 days). Quantitation of intestinal permeabilitywas carried out by quantitatively measuring plasma levels of endotoxinoriginating from gut bacteria using a commercially available chromogenicassay (Lonza, Rockland, Me.). The results of these experiments are shownin FIG. 1.

Quantitation of intestinal permeability can also be conducted using anumber of alternative methods (reviewed in Bischoff et al, 2014) forexample, by quantifying leakage of fluorescently-labeled high molecularweight dextran (FITC-dextran) into the plasma following oraladministration (oral gavage with 0.6 g/kg 4 kDa FITC-dextran, serumsamples collected 4 hours later and read for fluorescence intensity at521 nm; Hsiao et al, 2013). To study the effect of bacterial strains onintestinal permeability, mice are gavaged orally with 10⁷-10¹⁰ bacterialcells for an average of 5 administrations, typically daily or 2 daysapart. Bacteria can be administered as single strains or combinations ofstrains. The bacteria can be administered alone or in combination with apre-biotic(s). The pre-biotic can be xylose or xylose-containingmolecules as a preferred carbon source for anaerobic bacteria. Otherprebiotics that can be used include, for example, those described inTable 7. After administration of bacteria+/−pre-biotic, intestinalpermeability is assessed using the preferred method at the desired timepoint(s) starting on day 1 post-treatment.

As shown in FIG. 1, C57BL/6 mice were either left untreated or weretreated with xylose at 10 g/L in drinking water from day −7 to day 14;ciprofloxacin (cipro) at 0.25 g/L in drinking water from day −7 to day−2; enrofloxacin (enro) at 0.25 g/L in drinking water from day −7 to day−2; xylose+cipro or xylose+enro. Analysis of serum samples collected ondays 0 and 14 showed that basal levels of serum endotoxin are present innormal mice that remained unchanged in untreated mice. Xylose treatmentreduced these basal levels over time, suggesting an increase in gutbarrier integrity even in normal animals. Antibiotic treatment withcipro, a broad spectrum quinolone antibiotic, or enro, ananaerobe-sparing antibiotic, led to an increase in serum endotoxinlevels (measured 2 days after a 5 day course), likely due to disruptionof the microbiota. Serum endotoxin levels returned to baseline overtime. As shown in FIG. 1, xylose appeared to counteract the increase inserum endotoxin level caused by cipro, but not enro. The differentialeffect of xylose on these 2 antibiotics may relate to its ability topreserve/promote expansion of anaerobic bacteria, which are killed bycipro but not enro.

Example 2. Immunomodulatory Properties of Different Human CommensalBacteria on Human Peripheral Blood Mononuclear Cells

The microbiota of mammalian hosts is composed of bacterial species thatpossess both pro- and anti-inflammatory properties. In healthyindividuals, a balance or state of eubiosis is maintained that supportsgut barrier integrity, immune containment of commensal bacteria andpromotion of a tolerogenic environment. Under disease conditions,dysbiosis characterized by an imbalance in pro- and anti-inflammatorybacteria results in local inflammation and compromised gut barrierintegrity, leading to systemic inflammation and aberrant immuneresponses. Administration of selected probiotic bacterial strains(+/−prebiotics) that possess anti-inflammatory activity and promoteimmune tolerance represents an approach to correct a basic defectunderlying multiple pathological conditions.

An in vitro system was developed to efficiently test the inflammatoryand immunomodulatory properties of different human commensal bacteria onhuman peripheral blood mononuclear cells (PBMCs). Experiments werecarried out with 21 bacterial candidates to profile theiranti-inflammatory properties against human PBMCs. The innate propertiesof bacteria alone on human PBMCs were tested as well as their ability tocounteract the pro-inflammatory activity of Enterococcus feacalis.

Human PBMCs were isolated from fresh blood by density-gradientcentrifugation using Ficoll (1-4). Freshly isolated PBMCs were plated at1.5×10⁶ cells per ml per well of a 24-well plate in a total volume of 2mls RPMI-1640 medium+5% human serum, and incubated at 37° C./5% CO₂ withthe following:

-   -   (1) 500 □l of different commensal bacteria suspensions at OD 0.8    -   (2) E. faecalis at 10⁷ colony-forming units (cfu)    -   (3) A combination of commensal bacteria (OD 0.8)+E. Faecalis        (10⁷ cfu)    -   (4) Complete medium alone as a negative control    -   (5) Bacterial lipopolysaccharide (LPS; 100 ng/ml) as an        immunomodulatory “positive” control        Culture supernatants were collected at 24, 48 and 72 h, and the        cytokine profile was analyzed by Luminex technology according to        manufacturer's instruction (EMD Millipore, Danvers, Mass.).        Cytokine production was detectable in culture supernatants by 24        h with levels increasing over 48-72 h and sometimes exceeding        the range of quantitation. The results are presented in FIGS.        2-5 for all time points. The 24 h time point was chosen as the        optimal time point for further analysis. The 24 h results are        shown as a composite in FIG. 6 and with statistical analysis on        individual cytokines in FIGS. 7-10. The results represent the        properties of each bacterial species against human PBMCs and        their ability to counteract inflammatory stimulation with E.        faecalis in vitro. It was found that the commensal bacteria        tested have distinct immunomodulatory properties, and most        appear to counteract the inflammatory activity of E. Faecalis        for at least one cytokine.

FIG. 2 shows the time course of Th1 related cytokines that were releasedby human PBMCs incubated with Ruminococcus gnavus (Epv 1), Eubacteriumrectale (Epv 2), Blautia luti (Epv 3), Blautia wexlerae (Epv 5) andEnterococcus faecalis (Epv 8), or combinations of each bacterium with E.faecalis. Amounts of Th1-related pro-inflammatory cytokines interferongamma (IFN-γ), interleukin-12 p70 (IL-12p70), interleukin-6 (IL-6),interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFα) released byPBMCs were measured after 24, 48 and 72 hours. As shown in FIG. 2, allcommensals have unique immunomodulatory properties. As expected, E.faecalis induced high levels of these pro-inflammatory cytokines. Bycomparison, most of the other bacterial candidates induced lower levelsof Th1-related cytokines and were able to counteract the induction ofone or more inflammatory cytokines by E. faecalis. In particular,Blautia luti (Epv 3), showed minimal induction of Th1-related cytokineson its own and was most effective in counteracting induction of thesecytokines by E. faecalis (Epv 8). This profile is desirable for diseaseindications which are primarily driven by Th1 immune responses, such asGVHD.

FIG. 3 shows the time course of Th2 related cytokines that were releasedin cells treated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti(Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), or combinationsthereof. Amounts of anti-inflammatory cytokines interleukin-13 (IL-13),interleukin-4 (IL-4) and interleukin-5 (IL-5) released by PBMCs weremeasured after 24, 48 and 72 hours. Each bacterium displayed detectablepattern of cytokine induction and ability to modulate the effect of E.faecalis. Th2-related cytokines are beneficial in counteracting Th1responses. Bacteria capable of promoting Th2 cytokine release aretherefore of interest in Th1-driven diseases. R. gnavus appeared themost active in terms of eliciting Th2 cytokine on its own or in thepresence of E. faecalis.

FIG. 4 shows the time course of Th9, Th17 and Treg cytokines that werereleased in cells treated with R. gnavus (Epv 1), E. rectale (Epv 2), B.luti (Epv 3), B. wexlerae (Epv 5) and E. faecalis (Epv 8), orcombinations thereof. Amounts of interleukin-9 (IL-9), interleukin-17(IL-17) and interleukin-10 (IL-10) released by PBMCs were measured after24, 48 and 72 hours. The activity of IL-9 and IL-17 is context-dependentin that these cytokines can be beneficial under some conditions butdetrimental under other conditions depending on the mechanismsresponsible for disease pathogenesis. For example, IL-17 is expected tocontribute to disease pathogenesis in GVHD but could provide a benefitin Th2-driven disorders. IL-10 produced by regulatory T cells (Treg) isgenerally immunosuppressive and is expected to provide a benefit in mostinflammatory disorders whether Th1- or Th2-driven. As shown in FIG. 4,all bacterial candidates elicited IL-9 and IL-17 to varying degrees andB. wexlerae (Epv 5) was the most potent in inducing IL-10.

FIG. 5 shows the time course of monocyte, macrophage andneutrophil-related inflammatory cytokines that were released by PBMCstreated with R. gnavus (Epv 1), E. rectale (Epv 2), B. luti (Epv 3), B.wexlerae (Epv 5) and E. faecalis (Epv 8), or combinations thereof.Amounts of monocyte chemotactic protein 1 (MCP-1), macrophageinflammatory protein 113 (MIP1β), macrophage inflammatory protein 1α(MIP1α), regulated on activation, normal T expressed and secretedprotein (RANTES), interleukin-1α (IL-1α), interleukin-1β (IL1β),interferon α2 (IFN-α2) and interleukin-8 (IL-8) that were released weremeasured after 24, 48 and 72 hours. In general, these cytokinescontribute to inflammation by innate immune effector cells. The bacteriatested showed different degrees of induction and effects on E. faecalis.Overall, E. rectale (Epv 2) and B. luti (Epv 3) were the leastinflammatory and the most effective at countering the effect of E.faecalis (Epv 8).

A composite illustration of the secretion of each of thepro-inflammatory and anti-inflammatory cytokines described above in thepresence of each commensal alone or in combination with EPV8 is graphedrelative to the pro-inflammatory bacterial strain E. faecalis (Epv 8) inFIG. 6. In the context of GVHD, IFNγ (IFNg), IL-12p70, IL-1α (IL-1α),IL-6, IL-8, MCP1, MIP1α (MIP1α), MIP1β (MIP1b) and TNF (TNFα) areconsidered pro-inflammatory cytokines. IL-10, IL-13, IL-9, IL-4 and IL-5are considered anti-inflammatory cytokines. IL-17 (IL-17A), IL-9 andIL-2 have context dependent activity. The results are shown as apercentage of Epv 8, where cytokine levels in the presence of E.faecalis after 24 hours is set at 100%. Each commensal has a uniquesignature and each one added alone to human PBMCs appeared to be lessinflammatory than E. fecalis (below 100% for pro-inflammatorycytokines), except for B. wexlerae (Epv 5). When added to PBMCs incombination with E. faecalis, most commensals tested (except for Epv 5)also counteracted the pro-inflammatory activity of E. faecalis (below100% for pro-inflammatory cytokines).

FIGS. 7-10 detail individual cytokine profiles of PBMCs followingexposure to various commensals, alone or in combination with thepro-inflammatory bacterium E. faecalis (Epv8). In particular, FIG. 7shows the effect of R. gnavus (EPV1) on cytokine concentration (pg/ml)either alone or in combination with Epv 8 (E. faecalis).

FIG. 8 shows the effect of E. rectale (EPV 2) on cytokine concentration(pg/ml) either alone or in combination with Epv 8 (E. faecalis). FIG. 9shows the effect of B. luti (EPV 3) on cytokine concentration (pg/ml)either alone or in combination with Epv 8 (E. faecalis). FIG. 10 showsthe effect of B. wexlerae (EPV 5) on cytokine concentration (pg/ml)either alone or in combination with Epv 8 (E. faecalis).

Overall, the foregoing data indicate that, among the bacteria tested,EPV3 has a significantly desirable anti-inflammatory profile for aTh-1-driven condition, such as GVHD while EPV5 has a suboptimalanti-inflammatory profile for GVHD. As shown in FIG. 11, EPV3 hasrelatively low intrinsic inflammatory activity compared to EPV 8 and isable to reduce the induction of pro-inflammatory cytokines by EPV 8,including IL-6, MCP-1, IL-12p70, and IFNγ which are believed tocontribute to the pathogenesis of GVHD. By comparison, EPV 5 is similarto EPV 8 in terms of induction of pro-inflammatory cytokines and showslittle ability to counteract the induction of pro-inflammatory cytokinesby EPV 8.

Additional bacteria were profiled using this methodology including:Clostridium leptum (EPV 6), Blautia faecis (EPV15), Blautia/Ruminococcusobeum ATCC 29174 (EPV 20), Blautia product ATCC 27340 (EPV 21), Blautiacoccoides ATCC 29236 (EPV 22), Blautia hydrogenotrophica ATCC BAA-2371(EPV-23) and Blautia Hansenii ATCC27752 (EPV 24). Strains freshlyisolated by Epiva from the stool of a normal healthy volunteer were alsoprofiled and included: Eubacterium rectale (EPV 35), a previouslyuncultured Blautia, similar to GQ898099_s S1-5 (EPV 47), a previouslyuncultured Blautia, similar to SJTU_C_14_16 (EPV 51), Blautia wexlerae(SJTU_B_09_77) (EPV 52), Blautia luti ELU0087-T13-S-NI_000247 (EPV 54),Blautia wexlerae WAL 14507 (EPV 64), Blautia obeum (EPV 78),Ruminococcus gnavus (EPV 102) and Blautia luti (BlnIX) (EPV 114).Results focusing on key pro-inflammatory (IL-12p70, IFNγ, IP-10, IL-1RA)and anti-inflammatory (IL-10, IL-4, IL-13) cytokines are shown in FIGS.12-27. As observed with the initial set of bacterial candidates, eachisolate displayed a defined signature. Candidates for treatment ofautoimmune or inflammatory disorders, such as GVHD, displayed lowinduction of pro-inflammatory cytokines and/or positive induction ofanti-inflammatory cytokines, and had ability to counteract theinflammatory activity of E. faecalis. Bacterial candidates meeting thesecriteria include, for example, EPV 35, 51, 78 and 114.

Taken together, these results show that commensals have distinctimmunomodulatory properties and display a definable signature in termsof their ability to induce cytokines in human host cells, or counteractthe pro-inflammatory activity of another bacterium (E. faecalis).Accordingly, bacterial compositions may be selected in order to achievea desired modulation of pro- and anti-inflammatory cytokines. Forexample, anti-inflammatory bacterial strains may be selected based ontheir ability to reduce key pro-inflammatory cytokines such asinterferon gamma, IL-12p70, IP-10 and IL-1RA and/or increaseanti-inflammatory cytokines such as IL-13, IL-10 and IL-4.

Example 3. Effect of Commensal Human Bacteria on T-Cell Polarization

In order to determine whether exposure to commensal bacteria maypolarize T cells toward a particular phenotype, flow cytometry analysiswas performed on human PBMCs cultured with various commensal bacteria asdescribed above. The cells recovered from culture were washed inphosphate-buffered saline and stained with a cocktail of fluorescentlylabeled antibodies against specific cell surface protein markers toallow for the detection of Th1 cells (CXCR3⁺CCR6⁻), Th2 cells(CXCR3⁻CCR6⁻), Th17 cells (CXCR3⁻CCR6⁺) and Tregs (CD25⁺CD127^(lo)).Negative control wells contained PBMCs in culture medium alone andpositive control wells contained PBMCs+LPS (100 ng/ml) as a known immunestimulus. The commensal bacteria examined included: Epv 1: R. gnavus;Epv 3: B. luti; Epv 2: E. rectale; Epv 5: B. wexlerae; Epv. 8: E.faecalis; Epv 20: B. obeum, ATCC 29174; Epv 21: B. product, ATCC 27340;Epv 24: B. hansenii, ATCC 27752. As shown in FIG. 28, exposure of humanPBMCs to bacteria did result in a shift in the relative proportion of Tcell populations compared to the PBMCs alone (control) althoughstatistical significance was not achieved in every case. Overall, mostbacteria tested caused an increase in the proportion of T cells with aregulatory phenotype (Tregs) with EPV 21 and EPV 24 having the greatestimpact and EPV8 (E. faecalis) causing little or no increase in Tregs.Most bacteria also caused a decrease in the proportion of Th17 cells, anincrease in Th2 cells and had little or no effect on the proportion ofTh1 cells. This type of analysis indicates that commensal bacteria canmodulate the proportions of effector T cell types and can be used toselect the desired phenotype for a given disease application. Forexample, the optimal T cell profile to address pro-inflammatorydisorders such as GVHD would consist of ⇑Treg, ⇓Th17, ⇓ or unchangedTh1, and ⇑Th2. This phenotype was induced by many of the bacteriatested.

Example 4. Pattern of Carbon Source Utilization by Commensal Bacteria

Modulation of the microbiota to correct a dysbiosis associated withpathological conditions can potentially be achieved throughadministration of bacteria (or bacterial combinations) and prebiotic(s)as a carbon source to promote endogenous expansion of beneficialbacteria. Alternatively, prebiotics can be administered in combinationwith bacteria to promote their growth or create a favorable environmentfor their growth. Profiling of carbon source usage by bacterial isolatescan be used to customize and optimize selection of prebiotics forparticular bacterial strains. Profiling of carbon source usage wasconducted on 21 anaerobic commensal bacteria (Table 6) using 96 wellplates from Biolog (Hayward, Calif.) where each well contains adifferent carbon source for a total of 192 different carbon sources(PM01 and PM02A plates). The carbon sources tested are listed in Table7. The assay was conducted according to manufacturer's instructions.Briefly, pre-cultured bacteria were suspended in Biolog assay medium ata 750 nm optical density (OD) of 0.3-0.4, and 100 μl of the suspensionwas transferred to each well of the 96 well PM01 and PM02 assay plates.The plates were then incubated at 37° C. in an anaerobic chamber for 24hr or longer. The amount of growth on each carbon source was evaluatedby measuring the optical density (OD) of each well at 750 nm. Theresults are summarized in FIG. 29, and indicate that each individualstrain displays a unique pattern of carbon source usage. Interestingly,different isolates of the same species (e.g. B. luti and B. wexlerae)show related (albeit distinct) patterns. Overall, these results indicatethat characterization of carbon source usage for profiling of bacterialcandidates allows optimal selection of prebiotics. Preferred prebioticscan be selected which increase the growth (indicated by an increase inoptical density) of bacterial species contained in probioticcompositions.

Example 5. Normal Human Volunteer Study of a Prebiotic FormulationContaining Xylose

D-xylose is a carbon source generally preferred by anaerobic bacteria.Preliminary results in the mouse indicate that it may act to promote gutbarrier integrity (FIG. 1). It is also used as a carbon source byseveral bacterial strains (FIG. 29) that were determined to possess adesirable immunological profile for target indications such as GVHD(FIG. 19, 25, 27). A parallel, double-blind, 5 cohort escalation foodsafety study was conducted to examine D-xylose in normal humanvolunteers. The study was a double-blind, single-center, parallel groupstudy designed to evaluate the tolerability and potential microbiomechanges induced by ingestion of D-xylose at 5 different amounts inhealthy, adult volunteers enrolled at 1 study center in the UnitedStates (US).

Subjects were screened for eligibility within 21 days prior to the firstplanned ingestion of study sweetener on Day 1 (Baseline). Within each of5 cohorts, eligible subjects were randomly assigned in a double-blinded,6:2 ratio to ingest either D-xylose or the GRAS sweetener Splenda®(control), dissolved into 2 to 6 oz of sterile water and ingested TIDwith meals for a total of 82 ingestions taken over 28 consecutive days.D-xylose ingestion amounts ranged from 1 to 15 g TID (total daily amountof 3 to 45 g), and all subjects randomized to Splenda® ingested 1dissolved, commercially available packet TID (3 packets total per day).

Subjects returned to the study center weekly on Days 8, 15, 22, and 28for ingestion, tolerability, and compliance evaluations. Safety wasevaluated on a continual basis through adverse events (AE) monitoring,clinical laboratory measurements, vital sign monitoring, physicalexaminations, electrocardiograms (ECGs), telephone follow-up, andelectronic subject ingestion diaries. Stool was collected pre-ingestionand at pre-specified time points, and post-ingestion samples wereevaluated for changes in the gut microbiome compared with Baseline forall subjects. For subjects who consented to further sampling, additionalstool specimens were used to potentially isolate living bacteria thatcould be categorized for research and potential commercializationpurposes. Serum and urine were collected for measurement of D-xyloselevels and pharmacokinetic (PK) assessments and PK/pharmacodynamics (PD)correlations. Telephone follow-up was conducted as needed, but minimallyonce per week. The total duration for each participant was up to 60days, including the Screening period (Day −21 to 0), the ingestionperiod (Day 1 to 28), and an End-of-Study (EOS) follow-up visitconducted 7 (±3) days after the last ingestion of study sweetener.

Criteria for Evaluation

Safety

Safety was evaluated on a continual basis through AE monitoring,clinical laboratory measurements, vital sign monitoring, physicalexaminations, ECGs, telephone follow-up, and electronic subjectingestion diaries.

Immunology and Other Assessments

Stool was collected at pre-specified pre- and post-ingestion time pointsand post-ingestion samples were evaluated for changes in the gutmicrobiome compared with Baseline. Additional optional specimens werecollected to potentially isolate living bacteria that could becategorized for research and potential commercialization purposes.

Blood was collected at pre-specified pre- and post-ingestion time pointsto evaluate C-reactive protein (CRP), serum cytokines (tumor necrosisfactor alpha [TNF-α], interleukin [IL]-2, IL-6, interferon gamma[IFN-γ], and IL-10), and T-cell markers CD3, CD4, CD8, CD25, and FOXP3.Plasma was also stored and may be tested for biomarkers and/or metabolicmarkers for up to 7 years.

Pharmacokinetics

Blood and urine were collected at pre-specified pre- and post-ingestiontime points to measure D-xylose levels and to characterize the systemicabsorption profiles of D-xylose.

Statistical Methods

Statistical analyses were conducted using SAS®, Version 9.2 (SASInstitute, Inc., Cary, N.C., USA). The sample size calculations wereempiric and based on an estimation of normal healthy volunteervariability in reported symptoms and side effects and not on astatistical method. A weighted randomization scheme was implemented suchthat more subjects were enrolled at the higher D-xylose ingestionamounts to account for potential toxicity-related effects that couldhave resulted in withdrawal and/or analysis ineligibility, and to enablecollection of more data at ingestion amounts for which limited data wereavailable.

Analysis Populations

The safety population comprised all subjects who ingested any amount ofstudy sweetener.

Safety

AEs were coded using the Medical Dictionary for Regulatory Activities(MedDRA), Version 18.0 (Northrup Grumman Corporation, Chantilly, Va.,USA), and summarized by cohort. Laboratory, vital sign, and physicalexamination data were summarized by cohort using descriptive statisticsover time, including statistics for changes from Baseline. ECG findingswere also summarized by cohort over time as well as using frequencycounts and percentages, as normal or abnormal, with the relevance ofabnormalities categorized by clinical significance.

Immunology and Other Assessments

Stool sample compliance was summarized by cohort, using the followingcalculation for each subject:

${{Percentage}\mspace{14mu} {compliance}} = {\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{11mu} {stool}\mspace{14mu} {samples}\mspace{14mu} {collected}}{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {stool}\mspace{14mu} {samples}\mspace{14mu} {expected}} \times 100}$

A total of 7 stool samples were expected to be collected for eachsubject. Evaluation of changes in the gut microbiome were evaluated instool samples through taxonomic classification, relative and statisticaldifferential abundance analyses by cohort and time point, an alphadiversity analysis calculated using the Shannon diversity index bycohort and time point, a beta diversity analysis using Bray-Curtisdissimilarity and Unifrac distance by subject and time point, and aprincipal coordinates analysis using the beta diversity data.

Summary statistics (n, mean, standard deviation, median, minimum, andmaximum) were presented for serum concentrations of CRP, flow cytometryT-cell markers (CD3, CD4, CD8, CD25, and FOXP3), and cytokines (TNF-α,IL-2, IL-6, IFN-γ, and IL-10) as per their nominal time points.

Pharmacokinetics

Phoenix® WinNonLin®, Version 6.2.1, was used for PK analyses.

Serum D-xylose concentrations were summarized by cohort using nominalsample times according to actual amount received using summarystatistics (n, coefficient of variation [CV], mean, standard deviation[SD], median, minimum, and maximum). Evidence for the occurrence ofsteady-state was assessed graphically by comparing the time course ofeither trough or 2-hour post-ingestion serum concentrations of D-xyloseas different levels of D-xylose. Accumulation was assessed by comparingthe 2-hour post-first-ingestion serum levels with those observed at Week2 (Day 15) and Week 4 (Day 28).

The total amount of D-xylose excreted in urine was analyzed for allsubjects over 5 hours post-ingestion and pooled for analysis; thepooling for analysis reflected the subject mean within a given time ofcollection (e.g., Day 15 and then Day 28) sorted by ingested amount.Urine PK parameters for D-xylose levels included Ae_((0-t)) (cumulativeamount of sweetener recovered in urine) and percent sweetener amountexcreted over a 5-hour period.

Summary of Results

Forty-eight subjects were randomized to ingest either 1 packet ofcommercially-available Splenda® TID (n=12) or D-xylose TID at thefollowing ingestion amounts (n=36 total):

1 g: 6 subjects2 g: 6 subjects8 g: 7 subjects12.5 g: 8 subjects15 g: 9 subjects

Over the 28-day ingestion period, study sweetener ingestion compliancewas >90% for all subjects. Two subjects (4.2%) discontinued from thestudy prematurely; primary reasons for discontinuation were a protocolviolation (positive urine drug screen) and withdrawal of consent. Theproportion of males (47.9%) and females (52.1%) was balanced, and themajority of subjects were White (89.6%) and not Hispanic or Latino(77.1%). Subject ages spanned a wide range, with a median of 38.3 (range22.5 to 60.5) years for the combined D-xylose cohorts and 43.6 (range24.9 to 64.3) years for the Splenda® cohort.

Safety

D-xylose and Splenda® were both well tolerated, with no new safetyconcerns identified. One subject required a D-xylose reduction from 15 gto 12.5 g TID at the Week 1 (Day 8) visit due to AEs of moderateabdominal distension, diarrhea, and GI pain; no other modifications tosweetener ingestion amounts were implemented.

Overall, 17 subjects (35.4%) experienced at least 1 AE, including ahigher proportion of subjects who ingested any amount of D-xylose (14subjects [38.9%]) than Splenda® (3 subjects [25.0%]). Reported AE ratesincreased with increasing D-xylose ingestion amounts, with incidencesranging from 16.7% in subjects who ingested the 2 lowest amounts (1 and2 g TID) to 66.7% in subjects who ingested the highest amount (15 gTID). AEs reported for more than 1 subject in the D-xylose cohortsincluded diarrhea (3 subjects [8.3%]) and flatulence and GI pain (2subjects [5.6%] each). AEs in the Splenda® cohort included abdominaldistension, flatulence, increased blood creatinine, infrequent bowelmovements, and rhinitis. The incidence of AEs was highest during Weeks 1and 2 (Days 2 through 15), regardless of sweetener type or ingestionamount. During this 2-week period, 18 subjects overall (37.5%)experienced AEs, compared with 7 subjects (14.6%) overall whoexperienced AEs either on Day 1 or after Week 2.

All AEs were mild in severity with the exception of moderate AEsreported for 4 subjects (11.1%) in the D-xylose cohorts. These moderateAEs included abdominal distension, concussion/post-concussion syndrome,diarrhea, GI pain, increased blood bilirubin, and neutropenia.

No SAEs, severe AEs, or subject deaths were reported. One subject in the8 g TID D-xylose cohort experienced non-serious, moderate AEs ofconcussion and post-concussion syndrome that were noted to havecontributed to study discontinuation; however, this subject's primaryreason for discontinuation was withdrawal of consent.

GI-related AEs, which were of special interest, were reported for 7subjects (19.4%) in the D-xylose cohorts and 2 subjects (16.7%) in theSplenda® cohort. GI-related events were mild for all but 1 subject inthe 15 g TID D-xylose cohort who experienced moderate GI-related AEs ofabdominal distension, diarrhea, and GI pain that required reduction ofthe D-xylose ingestion amount to 12.5 g TID.

Eleven subjects (22.9%) experienced at least 1 AE that was considered bythe Investigator to be related to study sweetener, including 9 subjects(25.0%) in the D-xylose cohorts and 2 subjects (16.7%) in the Splenda®cohort. The incidence of sweetener-related AEs appeared to increase withincreasing D-xylose ingestion amounts. Sweetener-related AEs reportedfor more than 1 subject in the D-xylose cohorts included diarrhea (3subjects [8.3%]) and flatulence and GI pain (2 subjects [5.6%] each).Sweetener-related AEs reported in the Splenda® cohort were abdominaldistension, flatulence, and infrequent bowel movements.

No fluctuations in clinical laboratory measurements over time wereconsidered to be clinically meaningful. Categorical shifts from Baselinethat occurred in >10% of subjects in either the combined D-xylose orSplenda® cohorts included decreased or increased glucose (27.7% D-xyloseand 16.7% Splenda®) and decreased absolute neutrophil count (ANC) (13.9%and 8.3%); these shifts were not associated with sweetener type oringestion amount.

Immunology and Other Assessments

To assess the effect of D-xylose on the gut microbiome, this studyincorporated an analysis of alpha diversity, beta diversity, anddifferentially abundant taxa. These factors were assessed both acrosscohorts and over time. Regardless of sweetener ingestion amount, noapparent significant impact on the intra-sample alpha diversity of thegut microbiome was observed, and no significant changes in communitycomposition were observed over time on study. Numerous taxa wereidentified as differentially abundant, but these findings may reflectthe relatively small sample sizes in each cohort.

Across all D-xylose cohorts, 8.3% of subjects with normal serum CRP atBaseline experienced at least 1 post-ingestion CRP value >2.9 mg/L. Asubstantially higher proportion of subjects in the Splenda® cohort(41.7%) had normal serum CRP at Baseline and experienced at least 1post-ingestion CRP value >2.9 mg/L. None of the post-ingestion CRPvalues for any subject were deemed clinically significant.

Because most individual cytokine data points were below the limit ofquantitation (BLQ) and therefore set to zero, cytokine summarystatistics were limited and did not indicate any consistent orclinically meaningful changes over time for either sweetener or anyD-xylose ingestion amount. There was a trend for reduced levels of seruminterferon gamma over time in the 2 g and 15 g D-xylose cohorts (FIG.30). No consistent or clinically meaningful changes over time in totalT-cells or any T-cell subsets were observed for either sweetener or anyD-xylose ingestion amount.

Pharmacokinetics

Serum D-xylose concentrations increased linearly with increasingingestion amounts. Little to no accumulation of serum D-xylose occurredat Day 15 following 1 g to 12.5 g TID ingestion, while an approximately1.9-fold accumulation ratio was observed in the 15 mg TID cohort(although variability was high). On Day 28, the accumulation ratioranged from 1.08 to 1.31 following 1 g to 12.5 g TID ingestion and 1.68following 15 g TID ingestion, although variability was moderate to highin all but the 8 g TID cohort.

In the 1 g TID cohort, approximately 40% of the ingested amount ofD-xylose was recovered in urine within 5 hours post-ingestion on Days 1,15, and 28. In the 2 g through 15 g TID cohorts, between 23% and 32% ofthe ingested amount of D-xylose was recovered in urine within 5 hourspost-ingestion on Days 1, 15, and 28. The fraction excreted in urine wassimilar among Days 1, 15, and 28.

A review of the time course of serum D-xylose concentrations and thecorresponding urinary excretion profiles indicated high ingestioncompliance.

Changes in the Gut Microbiome

A total of 344 stool samples were collected in OMNIgene•GUT collectionkits and shipped to the GenoFIND laboratory for DNA extraction and V3-V416S amplicon sequencing. There were no major shifts in the microbiomealpha diversity between the different treatment groups (absolute numberof OTUs, abundance of OTUs) or over time on study. There was an overalldecrease in the Chao diversity index over time (indicator of communityrichness—# of singleton, doubleton OTUs), as shown in FIG. 31. Numeroustaxa were identified as differentially abundant, but this finding may beattributable to the relatively small sample sizes of each cohort.Similar observations were made in the mouse study, e.g., xylosetreatment did not cause major shifts in the gut microbiome but showedsome differences at the family level. Overall, these results suggestthat, under the conditions tested in normal individuals and normal mice,ingestion of xylose exerts subtle changes in the gut microbiome. Theimpact of xylose on the microbiome under disease conditions remains tobe determined.

Taken together, the results of this trial show that D-xylose is safe andwell-tolerated, and indicate that prebiotic formulations containingxylose may reduce inflammation in a subject, resulting in reduction ofserum levels of pro-inflammatory cytokines.

Example 6. Distal Augmentation

The trillions of organisms forming the microbiome function as an organsystem interconnected throughout the body. The possibility thatmodification of the microbiome in a given physical location mayinfluence the microbiome at other sites in the body (distalaugmentation) was investigated. Seven week old C57Bl/6 female mice wereacclimatized for 7 days prior to the start of the study by dailyhandling and shuffling between cages. All mice were housed at three miceper cage in individually vented cages (Thoren, Hazleton, Pa.). At day 0,baseline fresh fecal pellets, and vaginal lavages with 100 μL of steriledouble-distilled water were collected and immediately frozen at −80° C.for microbiome analysis. After baseline collection, mice were given todrink either autoclaved water (N=6) or 0.5 mg/L of the antibioticvancomycin in autoclaved water (N=6) ad libitum. Water alone is notexpected to influence the microbiome and acted as a negative control.Oral vancomycin is poorly absorbed from the gut and its ingestion doesnot result in significant levels of drug in the body (Rao et al, 2011).The impact of oral vancomycin is therefore expected to be limited to thegastrointestinal tract such that microbiome changes elsewhere in thebody (e.g. vagina) would be attributable to distal augmentation. At day6, fresh fecal pellets and vaginal lavages with 100 μL of steriledouble-distilled water were collected and immediately stored at −80° C.for microbiome analysis.

Isolation and sequencing of microbial DNA from the stool and vaginalsamples was performed by DNA Genotek (Ottawa, ON, Canada). The V3-V4region of the 16S ribosomal subunit was amplified with custom PCRprimers and sequenced on an Illumina MiSeq to a minimum acceptable readdepth of 25,000 sequences per sample. The widely accepted read depthrequirement for accurate taxonomic profiling is 15,000-100,000 reads(Illumina, 2014). A closed-reference taxonomic classification wasperformed, where each sequence was aligned to the SILVA referencedatabase, version 123. Sequences were aligned using the UCLUST algorithmincluded in QIIME version 1.9.1 (Caporaso et al., 2010). A minimumthreshold of 97% sequence identity was used to classify sequencesaccording to representative sequences in the database. At 97% sequenceidentity, each OTU represents a genetically unique group of biologicalorganisms. These OTU's were then assigned a curated taxonomic labelbased on the seven level SILVA taxonomy.

As expected, oral vancomycin treatment had a strong impact on themicrobiome of the gut. As shown by principal component analysis (PCA) atthe family level, the day 0 to day 6 pattern in fecal samples wasclearly different in the control vs oral vancomycin group (FIG. 32).Interestingly, the day 0 to day 6 pattern in the vaginal samples alsoshowed an overall difference between the PBS and oral vancomycin groupseven though the vaginal environment is not exposed to vancomycinfollowing oral administration of the antibiotic (FIG. 32). In addition,some bacterial species were detected at low frequency in vaginal samplesof the vancomycin-treated group at day 6 (median abundance ofapproximately 0.00002%) that were not present at day 0 (Table 8). Theseresults support the concept of distal augmentation whereby modificationof the microbiome at one site also has an impact at a distal site(s).This finding opens the possibility of modulating the microbiome, forexample at the level of the gut, to effect therapeutic changes in themicrobiome at other sites, for example the lung.

Example 7. Provision of Fecal Material

Fresh fecal samples are obtained from healthy human donors who have beenscreened for general good health and for the absence of infectiousdiseases, and meet inclusion and exclusion criteria, inclusion criteriainclude being in good general health, without significant medicalhistory, physical examination findings, or clinical laboratoryabnormalities, regular bowel movements with stool appearance typicallyType 2, 3, 4, 5 or 6 on the Bristol Stool Scale, and having a BMI≧18kg/m² and ≦25 kg/m². Exclusion criteria generally include significantchronic or acute medical conditions including renal, hepatic, pulmonary,gastrointestinal, cardiovascular, genitourinary, endocrine, immunologic,metabolic, neurologic or hematological disease, a family history of,inflammatory bowel disease including Crohn's disease and ulcerativecolitis, Irritable bowel syndrome, colon, stomach or othergastrointestinal malignancies, or gastrointestinal polyposis syndromes,or recent use of yogurt or commercial probiotic materials in which anorganism(s) is a primary component. Samples are collected directly usinga commode specimen collection system, which contains a plastic supportplaced on the toilet seat and a collection container that rests on thesupport. Feces are deposited into the container, and the lid is thenplaced on the container and sealed tightly. The sample is then deliveredon ice within 1-4 hours for processing. Samples are mixed with a steriledisposable tool, and 2-4 g aliquots are weighed and placed into tubesand flash frozen in a dry ice/ethanol bath. Aliquots are frozen at −80degrees Celsius until use.

Optionally, the fecal material is suspended in a solution, and/orfibrous and/or particulate materials are removed. A frozen aliquotcontaining a known weight of feces is removed from storage at −80degrees Celsius and allowed to thaw at room temperature. Sterile 1×PBSis added to create a 10% w/v suspension, and vigorous vortexing isperformed to suspend the fecal material until the material appearedhomogeneous. The material is then left to sit for 10 minutes at roomtemperature to sediment fibrous and particulate matter. The suspensionabove the sediment is then carefully removed into a new tube andcontains a purified spore population. Optionally, the suspension is thencentrifuged at a low speed, e.g., 1000×g, for 5 minutes to pelletparticulate matter including fibers. The pellet is discarded and thesupernatant, which contained vegetative organisms and spores, is removedinto a new tube. The supernatant is then centrifuged at 6000×g for 10minutes to pellet the vegetative organisms and spores. The pellet isthen resuspended in 1×PBS with vigorous vortexing until the materialappears homogenous.

Example 8. Spore Purification from Alcohol Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS is mixed withabsolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. Thesuspension is incubated at 37 degrees Celsius for 1 hour. Afterincubation the suspension is centrifuged at 13,000 rpm for 5 minutes topellet spores. The supernatant is discarded and the pellet isresuspended in an equal volume of PBS. Glycerol is added to a finalconcentration of 15% and then the purified spore fraction is stored at−80 degrees Celsius.

Example 9. Generation of a Spore Preparation from Alcohol Treatment ofFecal Material

A 10% w/v suspension of human fecal material in PBS is mixed withabsolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. Thesuspension is incubated at 37 degrees Celsius for 1 hour. Afterincubation the suspension is centrifuged at 13,000 rpm for 5 minutes toconcentrate spores into a pellet containing a purified spore-containingpreparation. The supernatant is discarded and the pellet resuspended inan equal volume of PBS. Glycerol is added to a final concentration of15% and then the purified spore preparation is stored at −80 degreesCelsius.

Example 10. Spore Purification from Thermal Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS is incubated in awater bath at 80 degrees Celsius for 30 minutes. Glycerol is added to afinal concentration of 15% and then the enriched spore containingmaterial is stored at −80 degrees Celsius.

Example 11. Spore Purification from Alcohol Treatment and ThermalTreatment of Fecal Material

A 10% w/v suspension of human feces in PBS is mixed with absoluteethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspensionis incubated in a water bath under aerobic conditions at 37 degreesCelsius for 1 hour. After incubation the suspension is centrifuged at13,000 rpm for 5 minutes to pellet spores. The supernatant is discardedand the pellet is resuspended in equal volume PBS. The ethanol treatedspore population is then incubated in a water bath at 80 degrees Celsiusfor 30 minutes. Glycerol is added to a final concentration of 15% andthe purified spore fraction is stored at −80 C.

Example 12. Construction of Binary and Ternary Combinations in aHigh-Throughput 96-Well Format

To allow high-throughput screening of binary and ternary combinations,vials of −80° C. glycerol stock banks are thawed and diluted to 1 e8CFU/mL. Each strain is then diluted 10× (to a final concentration of 1e7 CFU/mL of each strain) into 200 uL of PBS+15% glycerol in the wellsof a 96-well plate. Plates are then frozen at −80° C. When needed forthe assay, plates are removed from −80° C. and thawed at roomtemperature under anaerobic conditions when testing in a plate assaywith various pathogens.

Example 13. Spore Purification from Detergent Treatment of FecalMaterial

A 10% w/v suspension of human feces in PBS is prepared to contain afinal concentration of 0.5 to 2% Triton X-100. After shaking incubationfor 30 minutes at 25 to 37 degrees Celsius, the sample is centrifuged at1000 g for 5-10 minutes to pellet particulate matter and large cells.The bacterial entities are recovered in the supernatant fraction, wherethe purified spore population is optionally further treated, such as inExample 11. Without being bound by theory, detergent addition to thefecal mixture produces better spore populations, at least in part byenhancing separation of the spores from particulates thereby resultingin higher yields of spores. In some embodiments, the purified sporepopulation is further treated, such as by thermal treatment and/orethanol treatment as described above.

Example 14. Spore Purification by Chromatographic Separation of FecalMaterial

A spore-enriched population such as obtained from Examples 7-12 above,is mixed with NaCl to a final concentration of 4M total salt andcontacted with octyl Sepharose 4 Fast Flow to bind the hydrophobic sporefraction. The resin is washed with 4M NaCl to remove less hydrophobiccomponents, and the spores are eluted with distilled water, and thedesired enriched spore fraction is collected via UV absorbance.

Example 15. Spore Purification by Filtration of Fecal Material

A spore-enriched population such as obtained from Examples 8-13 above isdiluted 1:10 with PBS, and placed in the reservoir vessel of atangential flow microfiltration system. A 0.2 μm pore size mixedcellulose ester hydrophilic tangential flow filter is connected to thereservoir such as by a tubing loop. The diluted spore preparation isrecirculated through the loop by pumping, and the pressure gradientacross the walls of the microfilter forces the supernatant liquidthrough the filter pores. By appropriate selection of the filter poresize the desired bacterial entities are retained, while smallercontaminants such as cellular debris, and other contaminants in fecessuch as bacteriophage pass through the filter. Fresh PBS buffer is addedto the reservoir periodically to enhance the washout of thecontaminants. At the end of the diafiltration, the spores areconcentrated approximately ten-fold to the original concentration. Thepurified spores are collected from the reservoir and stored as providedherein.

Example 16. Characterization of Purified Spore Populations

Counts of viable spores are determined by performing 10 fold serialdilutions in PBS and plating to Brucella Blood Agar Petri plates orapplicable solid media. Plates are incubated at 37 degrees Celsius for 2days. Colonies are counted from a dilution plate with 50-400 coloniesand used to back-calculate the number of viable spores in thepopulation. The ability to germinate into vegetative bacteria is alsodemonstrated. Visual counts are determined by phase contrast microscopy.A spore preparation is either diluted in PBS or concentrated bycentrifugation, and a 5 microliter aliquot is placed into a PetroffHauser counting chamber for visualization at 400× magnification. Sporesare counted within ten 0.05 mm×0.05 mm grids and an average spore countper grid is determined and used to calculate a spore count per ml ofpreparation. Lipopolysaccharide (LPS) reduction in purified sporepopulations is measured using a Limulus amebocyte lysate (LAL) assaysuch as the commercially available ToxinSensor™ Chromogenic LALEndotoxin Assay Kit (GenScript, Piscataway, N.J.) or other standardmethods known to those skilled in the art.

Example 17. Quantification of C. difficile Using Quantitative PCR (qPCR)

A. Standard Curve Preparation

The standard curve is generated from a well on each assay platecontaining only pathogenic C. difficile grown in SweetB+FosIn media asprovided herein and quantified by selective spot plating. Serialdilutions of the culture are performed in sterile phosphate-bufferedsaline. Genomic DNA is extracted from the standard curve samples alongwith the other wells.

B. Genomic DNA Extraction

Genomic DNA is extracted from 5 μl of each sample using a dilution,freeze/thaw, and heat lysis protocol. 5 μL of thawed samples are addedto 45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) andmixed by pipetting. The plates with diluted samples are frozen at −20°C. until use for qPCR which includes a heated lysis step prior toamplification. Alternatively the genomic DNA could be isolated using theMo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo BioLabtoatories, Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (MoBio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

C. qPCR Composition and Conditions

The qPCR reaction mixture contained 1× SsoAdvanced Universal ProbesSupermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG,IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer(CATGCTTTTTTAGTTTCTGGATTGAA, IDT, Coralville, Iowa), 250 nM of Wr-tcdB-Pprobe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB, Life Technologies, GrandIsland, N.Y.), and Molecular Biology Grade Water (Mo Bio Laboratories,Carlsbad, Calif.) to 18 μl (Primers adapted from: Wroblewski, D. et al.,Rapid Molecular Characterization of Clostridium difficile and Assessmentof Populations of C. difficile in Stool Specimens, Journal of ClinicalMicrobiology 47:2142-2148 (2009)). This reaction mixture is aliquoted towells of a Hard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate(BioRad, Hercules, Calif.). To this reaction mixture, 2 of diluted,frozen, and thawed samples are added and the plate sealed with aMicroseal ‘B’ Adhesive Seal (BioRad, Hercules, Calif.). The qPCR isperformed on a BioRad C1000™ Thermal Cycler equipped with a CFX96™Real-Time System (BioRad, Hercules, Calif.). The thermocyclingconditions are 95° C. for 15 minutes followed by 45 cycles of 95° C. for5 seconds, 60° C. for 30 seconds, and fluorescent readings of the FAMchannel. Alternatively, the qPCR could be performed with other standardmethods known to those skilled in the art.

Example 18. 16S Sequencing to Determine Operational Taxonomic Unit (OTU)Method for Determining 16S Sequence

OTUs may be defined either by full 16S sequencing of the rDNA gene, bysequencing of a specific hypervariable region of this gene (i.e. V1, V2,V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination ofhypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial16S rDNA is approximately 1500 nucleotides in length and is used inreconstructing the evolutionary relationships and sequence similarity ofone bacterial isolate to another using phylogenetic approaches. 16Ssequences are used for phylogenetic reconstruction as they are ingeneral highly conserved, but contain specific hypervariable regionsthat harbor sufficient nucleotide diversity to differentiate genera andspecies of most microbes.

rDNA gene sequencing methods are applicable to both the analysis ofnon-enriched samples, but also for identification of microbes afterenrichment steps that either enrich the microbes of interest from themicrobial composition and/or the nucleic acids that harbor theappropriate rDNA gene sequences as described below. For example,enrichment treatments prior to 16S rDNA gene characterization willincrease the sensitivity of 16S as well as other molecular-basedcharacterization nucleic acid purified from the microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S rRNA sequence,genomic DNA is extracted from a bacterial sample, the 16S rDNA (fullregion or specific hypervariable regions) amplified using polymerasechain reaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingmay be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

Method for Determining 18S rDNA and ITS Gene Sequence

Methods to assign and identify fungal OTUs by genetic means can beaccomplished by analyzing 18S sequences and the internal transcribedspacer (ITS). The rRNA of fungi that forms the core of the ribosome istranscribed as a signal gene and consists of the 8S, 5.8S and 28Sregions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28Sregions, respectively. These two intercistronic segments between the 18Sand 5.8S and 5.8S and 28S regions are removed by splicing and containsignificant variation between species for barcoding purposes aspreviously described (Schoch et al Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker forFungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used forphylogenetic reconstruction however the ITS can serve this function asit is generally highly conserved but contains hypervariable regions thatharbor sufficient nucleotide diversity to differentiate genera andspecies of most fungus.

Using well known techniques, in order to determine the full 18S and ITSsequences or a smaller hypervariable section of these sequences, genomicDNA is extracted from a microbial sample, the rDNA amplified usingpolymerase chain reaction (PCR), the PCR products cleaned, andnucleotide sequences delineated to determine the genetic compositionrDNA gene or subdomain of the gene. The sequencing method used may be,but is not limited to, Sanger sequencing or using a next-generationsequencing method, such as an Illumina (sequencing by synthesis) methodusing barcoded primers allowing for multiplex reactions.

Method for Determining Other Marker Gene Sequences

In addition to the 16S rRNA gene, one may define an OTU by sequencing aselected set of genes that are known to be marker genes for a givenspecies or taxonomic group of OTUs. These genes may alternatively beassayed using a PCR-based screening strategy. As example, variousstrains of pathogenic Escherichia coli can be distinguished using DNAsfrom the genes that encode heat-labile (LTI, LTIIa, and LTIIb) andheat-stable (STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1,VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 andCNF2), attaching and effacing mechanisms (eaeA), enteroaggregativemechanisms (Eagg), and enteroinvasive mechanisms (Einv). The optimalgenes to utilize for taxonomic assignment of OTUs by use of marker geneswill be familiar to one with ordinary skill of the art of sequence basedtaxonomic identification.

Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hotalkaline lysis method. 1 μl of microbial culture is added to 9 μl ofLysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. andneutralized by the addition of 10 μl of Neutralization Buffer (40 mMTris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).Alternatively, genomic DNA is extracted from pure microbial culturesusing commercially available kits such as the Mo Bio Ultraclean®Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) orby standard methods known to those skilled in the art. For fungalsamples, DNA extraction can be performed by methods described previously(US20120135127) for producing lysates from fungal fruiting bodies bymechanical grinding methods.

Amplification of 16S Sequences for Downstream Sanger Sequencing

To amplify bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20μl final volume PCR reaction. For full-length 16 sequencing the PCRreaction also contains 1× HotMasterMix (5PRIME, Gaithersburg, Md.), 250nM of 27f (AGRGTTTGATCMTGGCTCAG, IDT, Coralville, Iowa), and 250 nM of1492r (TACGGYTACCTTGTTAYGACTT, IDT, Coralville, Iowa), with PCR Water(Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the volume.Alternatively, other universal bacterial primers or thermostablepolymerases known to those skilled in the art are used. For exampleprimers are available to those skilled in the art for the sequencing ofthe “V1-V9 regions” of the 16S rRNA. These regions refer to the firstthrough ninth hypervariable regions of the 16S rRNA gene that are usedfor genetic typing of bacterial samples. These regions in bacteria aredefined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879,986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. Brosius et al.,Complete nucleotide sequence of a 16S ribosomal RNA gene fromEscherichia coli, PNAS 75(10):4801-4805 (1978).

In some embodiments, OTUs may be defined either by full 16S sequencingof the rRNA gene, by sequencing of a specific hypervariable region ofthis gene (i.e., VI, V2, V3, V4, V5, V6, V7, V8, or V9), or bysequencing any combination of hypervariable regions from this gene(e.g., V1-3 or V3-5). In one embodiment, the VI, V2, and V3 regions areused to characterize an OTU. In another embodiment, the V3, V4, and V5regions are used to characterize an OTU. In another embodiment, the V4region is used to characterize an OTU. A person of ordinary skill in theart can identify the specific hypervariable regions of a candidate 16SrRNA by comparing the candidate sequence in question to the referencesequence and identifying the hypervariable regions based on similarityto the reference hypervariable regions.

The PCR is performed on commercially available thermocyclers such as aBioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). Thereactions are run at 94° C. for 2 minutes followed by 30 cycles of 94°C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30seconds, followed by a 7 minute extension at 72° C. and an indefinitehold at 4° C. Following PCR, gel electrophoresis of a portion of thereaction products is used to confirm successful amplification of a ˜1.5kb product.

To remove nucleotides and oligonucleotides from the PCR products, 2 μlof HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 5 μl ofPCR product followed by a 15 minute incubation at 37° C. and then a 15minute inactivation at 80° C.

Amplification of 16S Sequences for Downstream Characterization byMassively Parallel Sequencing Technologies

Amplification performed for downstream sequencing by short readtechnologies such as Illumina require amplification using primers knownto those skilled in the art that additionally include a sequence-basedbarcoded tag. As example, to amplify the 16s hypervariable region V4region of bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20 μlfinal volume PCR reaction. The PCR reaction also contains 1×HotMasterMix (5PRIME, Gaithersburg, Md.), 200 nM of V4_515f_adapt(AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCG GTAA, IDT,Coralville, Iowa), and 200 nM of barcoded 806rbc(CAAGCAGAAGACGGCATACGAGAT_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT, IDT, Coralville, Iowa), with PCR Water (Mo BioLaboratories, Carlsbad, Calif.) for the balance of the volume. Theseprimers incorporate barcoded adapters for Illumina sequencing bysynthesis. Optionally, identical replicate, triplicate, or quadruplicatereactions may be performed. Alternatively other universal bacterialprimers or thermostable polymerases known to those skilled in the artare used to obtain different amplification and sequencing error rates aswell as results on alternative sequencing technologies.

The PCR amplification is performed on commercially availablethermocyclers such as a BioRad MyCycler™ Thermal Cycler (BioRad,Hercules, Calif.). The reactions are run at 94° C. for 3 minutesfollowed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute, and72° C. for 1 minute 30 seconds, followed by a 10 minute extension at 72°C. and a indefinite hold at 4° C. Following PCR, gel electrophoresis ofa portion of the reaction products is used to confirm successfulamplification of a ˜1.5 kb product. PCR cleanup is performed asspecified in the previous example.

Sanger Sequencing of Target Amplicons from Pure Homogeneous Samples

To detect nucleic acids for each sample, two sequencing reactions areperformed to generate a forward and reverse sequencing read. Forfull-length 16s sequencing primers 27f and 1492r are used. 40 ng ofExoSap-IT-cleaned PCR products are mixed with 25 pmol of sequencingprimer and Mo Bio Molecular Biology Grade Water (Mo Bio Laboratories,Carlsbad, Calif.) to 15 μl total volume. This reaction is submitted to acommercial sequencing organization such as Genewiz (South Plainfield,N.J.) for Sanger sequencing.

In order to determine the full 16S sequence or the sequence of anyhypervariable region of the 16S rRNA sequence, genomic DNA is extractedfrom a bacterial sample, the 16S rDNA (full region or specifichypervariable regions) is amplified using polymerase chain reaction(PCR), the PCR products are cleaned, and nucleotide sequences delineatedto determine the genetic composition of 16S gene or subdomain of thegene. If full 16S sequencing is performed, the sequencing method usedmay be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4-V5 region, the sequencingmay be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

Amplification of 18S and ITS Regions for Downstream Sequencing andCharacterization

To amplify the 18S or ITS regions, 2 μL fungal DNA were amplified in afinal volume of 30 uL with 15 μL AmpliTaq Gold 360 Mastermix, PCRprimers, and water. The forward and reverse primers for PCR of the ITSregion are 5′-TCCTCCGCTTATTGATATGC-3′ and 5′-GGAAGTAAAAGTCGTAACAAGG-3′and are added at 0.2 uM concentration each. The forward and reverseprimers for the 18S region are 5′-GTAGTCATATATGCTTGTCTC-3′ and5′-CTTCCGTCAATTCCTTTAAG-3′ and are added at 0.4 uM concentration each.PCR is performed with the following protocol: 95 C for 10 min, 35 cyclesof 95 C for 15 seconds, 52 C for 30 seconds, 72C for 1.5s; and finally72C for 7 minutes followed by storage at 4C. All forward primerscontained the M13F-20 sequencing primer, and reverse primers includedthe M13R-27 sequencing primer. PCR products (3 μL) were enzymaticallycleaned before cycle sequencing with 1 μL ExoSap-IT and 1 μL Tris EDTAand incubated at 37° C. for 20 min followed by 80° C. for 15 min. Cyclesequencing reactions contained 5 μL cleaned PCR product, 2 μL BigDyeTerminator v3.1 Ready Reaction Mix, 1 μL 5× Sequencing Buffer, 1.6 pmolof appropriate sequencing primers designed by one skilled in the art,and water in a final volume of 10 μL. The standard cycle sequencingprotocol is 27 cycles of 10 s at 96° C., 5 s at 50° C., 4 min at 60° C.,and hold at 4° C. Sequencing cleaning is performed with the BigDyeXTerminator Purification Kit as recommended by the manufacturer for10-μL volumes. The genetic sequence of the resulting 18S and ITSsequences is performed using methods familiar to one with ordinary skillin the art using either Sanger sequencing technology or next-generationsequencing technologies such as but not limited to Illumina.

Preparation of Extracted Nucleic Acids for Metagenomic Characterizationby Massively Parallel Sequencing Technologies

Extracted nucleic acids (DNA or RNA) are purified and prepared bydownstream sequencing using standard methods familiar to one withordinary skill in the art and as described by the sequencingtechnology's manufactures instructions for library preparation. Inshort, RNA or DNA are purified using standard purification kits such asbut not limited to Qiagen's RNeasy Kit or Promega's Genomic DNApurification kit. For RNA, the RNA is converted to cDNA prior tosequence library construction. Following purification of nucleic acids,RNA is converted to cDNA using reverse transcription technology such asbut not limited to Nugen Ovation RNA-Seq System or Illumina Truseq asper the manufacturer's instructions. Extracted DNA or transcribed cDNAare sheared using physical (e.g., Hydroshear), acoustic (e.g., Covaris),or molecular (e.g., Nextera) technologies and then size selected as perthe sequencing technologies manufacturer's recommendations. Followingsize selection, nucleic acids are prepared for sequencing as per themanufacturer's instructions for sample indexing and sequencing adapterligation using methods familiar to one with ordinary skill in the art ofgenomic sequencing.

Massively Parallel Sequencing of Target Amplicons from HeterogeneousSamples

DNA Quantification & Library Construction.

The cleaned PCR amplification products are quantified using theQuant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island,N.Y.) according to the manufacturer's instructions. Followingquantification, the barcoded cleaned PCR products are combined such thateach distinct PCR product is at an equimolar ratio to create a preparedIllumina library.

Nucleic Acid Detection.

The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers(Illumina, San Diego, Calif.) with cluster generation, templatehybridization, isothermal amplification, linearization, blocking anddenaturation and hybridization of the sequencing primers performedaccording to the manufacturer's instructions. 16SV4SeqFw(TATGGTAATTGTGTGCCAGCMGCCGCGGTAA), 16SV4SeqRev(AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT), and 16SV4Index(ATTAGAWACCCBDGTAGTCCGGCTGACTGACT) (IDT, Coralville, Iowa) are used forsequencing. Other sequencing technologies can be used such as but notlimited to 454, Pacific Biosciences, Helicos, Ion Torrent, and Nanoporeusing protocols that are standard to someone skilled in the art ofgenomic sequencing.

Example 19. Data Analysis, Sequence Annotation and TaxonomicCharacterization Primary Read Annotation

Nucleic acid sequences are analyzed to define taxonomic assignmentsusing sequence similarity and phylogenetic placement methods or acombination of the two strategies. A similar approach is used toannotate protein names, protein function, transcription factor names,and any other classification schema for nucleic acid sequences. Sequencesimilarity based methods include those familiar to individuals skilledin the art including, but not limited to, BLAST, BLASTx, tBLASTn,tBLASTx, RDP-classifier, DNAclust, and various implementations of thesealgorithms such as Qiime or Mothur. These methods rely on mapping asequence read to a reference database and selecting the match with thebest score and e-value. Common databases include, but are not limited tothe Human Microbiome Project, NCBI non-redundant database, Greengenes,RDP, and Silva for taxonomic assignments. For functional assignmentsreads are mapped to various functional databases such as but not limitedto COG, KEGG, BioCyc, and MetaCyc. Further functional annotations can bederived from 16S taxonomic annotations using programs such as PICRUST(M. Langille, et al 2013. Nature Biotechnology 31, 814-821).Phylogenetic methods can be used in combination with sequence similaritymethods to improve the calling accuracy of an annotation or taxonomicassignment. Here tree topologies and nodal structure are used to refinethe resolution of the analysis. In this approach we analyze nucleic acidsequences using one of numerous sequence similarity approaches andleverage phylogenetic methods that are well known to those skilled inthe art, including but not limited to maximum likelihood phylogeneticreconstruction (see e.g. Liu K, Linder C R, and Warnow T. 2011. RA×MLand FastTree: Comparing Two Methods for Large-Scale Maximum LikelihoodPhylogeny Estimation. PLoS ONE 6: e27731. McGuire G, Denham M C, andBalding D J. 2001. Models of sequence evolution for DNA sequencescontaining gaps. Mol. Biol. Evol 18: 481-490. Wróbel B. 2008.Statistical measures of uncertainty for branches in phylogenetic treesinferred from molecular sequences by using model-based methods. J. Appl.Genet. 49: 49-67.) Sequence reads are placed into a reference phylogenycomprised of appropriate reference sequences. Annotations are made basedon the placement of the read in the phylogenetic tree. The certainty orsignificance of the OTU annotation is defined based on the OTU'ssequence similarity to a reference nucleic acid sequence and theproximity of the OTU sequence relative to one or more referencesequences in the phylogeny. As an example, the specificity of ataxonomic assignment is defined with confidence at the the level ofFamily, Genus, Species, or Strain with the confidence determined basedon the position of bootstrap supported branches in the referencephylogenetic tree relative to the placement of the OTU sequence beinginterrogated. Nucleic acid sequences can be assigned functionalannotations using the methods described above.

In some embodiments, microbial clades are assigned using databasesincluding, but not limited to, MetaPhlAn. Microbial diversity isquantified using the Shannon diversity index following closed-referenceoperational taxonomic unit picking. Phylogenetic abundance comparisonsare performed in order to identify biomarkers of GVHD-related mortalityusing linear discriminant analysis (LDA) effect size (LEfSe) analysis,using a logarithmic LDA cutoff of 2.0.

Clade Assignments

The ability of 16S-V4 OTU identification to assign an OTU as a specificspecies depends in part on the resolving power of the 16S-V4 region ofthe 16S gene for a particular species or group of species. Both thedensity of available reference 16S sequences for different regions ofthe tree as well as the inherent variability in the 16S gene betweendifferent species will determine the definitiveness of a taxonomicannotation. Given the topological nature of a phylogenetic tree and thefact that tree represents hierarchical relationships of OTUs to oneanother based on their sequence similarity and an underlyingevolutionary model, taxonomic annotations of a read can be rolled up toa higher level using a clade-based assignment procedure (Table 1). Usingthis approach, clades are defined based on the topology of aphylogenetic tree that is constructed from full-length 16S sequencesusing maximum likelihood or other phylogenetic models familiar toindividuals with ordinary skill in the art of phylogenetics. Clades areconstructed to ensure that all OTUs in a given clade are: (i) within aspecified number of bootstrap supported nodes from one another(generally, 1-5 bootstraps), and (ii) share a defined percentsimilarity, e.g., within a 5% genetic similarity (for 16S molecular datatypically set to 95%-97% sequence similarity). OTUs that are within thesame clade can be distinguished as genetically and phylogeneticallydistinct from OTUs in a different clade based on 16S-V4 sequence data.OTUs falling within the same clade are evolutionarily closely relatedand may or may not be distinguishable from one another using 16S-V4sequence data. The power of clade based analysis is that members of thesame clade, due to their evolutionary relatedness, are likely to playsimilar functional roles in a microbial ecology such as that found inthe human gut or vagina. Compositions substituting one species withanother from the same clade are likely to have conserved ecologicalfunction and therefore are useful in the present invention. Notably inaddition to 16S-V4 sequences, clade-based analysis can be used toanalyze 18S, ITS, and other genetic sequences.

Notably, 16S sequences of isolates of a given OTU are phylogeneticallyplaced within their respective clades, sometimes in conflict with themicrobiological-based assignment of species and genus that may havepreceded 16S-based assignment. Discrepancies between taxonomicassignment based on microbiological characteristics versus geneticsequencing are known to exist from the literature.

For a given network ecology or functional network ecology one can definea set of OTUs from the network's representative clades.

Metagenomic Read Annotation

Metagenomic or whole genome shotgun sequence data is annotated asdescribed above, with the additional step that sequences are eitherclustered or assembled prior to annotation. Following sequencecharacterization as described above, sequence reads are demultiplexedusing the indexing (i.e. barcodes). Following demultiplexing sequencereads are either: (i) clustered using a rapid clustering algorithm suchas but not limited to UCLUST(http://drive5.com/usearch/manual/uclust_algo.html) or hash methods suchVICUNA (Xiao Yang, Patrick Charlebois, Sante Gnerre, Matthew G Coole,Niall J. Lennon, Joshua Z. Levin, James Qu, Elizabeth M. Ryan, MichaelC. Zody, and Matthew R. Henn. 2012. De novo assembly of highly diverseviral populations. BMC Genomics 13:475). Following clustering arepresentative read for each cluster is identified based and analyzed asdescribed above in “Primary Read Annotation”. The result of the primaryannotation is then applied to all reads in a given cluster. (ii) Asecond strategy for metagenomic sequence analysis is genome assemblyfollowed by annotation of genomic assemblies using a platform such asbut not limited to MetAMOS (T J. Treangen et al. 2013 Geneome Biology14:R2), HUMAaN (Abubucker S. Segata N, Goll J, Schubert A M, Izard J,Cantarel B L, Rodriguez-Mueller B, Zucker J, Thiagarajan M, Henrissat B,et al. 2012. Metabolic Reconstruction for Metagenomic Data and ItsApplication to the Human Microbiome ed. J. A. Eisen. PLoS ComputationalBiology 8: e1002358) and other methods familiar to one with ordinaryskill in the art.

Example 20. OTU Identification Using Microbial Culturing Techniques

The identity of the bacterial species which grew up from a complexfraction can be determined in multiple ways. First, individual coloniescan be picked into liquid media in a 96 well format, grown up and savedas 15% glycerol stocks at −80° C. Aliquots of the cultures can be placedinto cell lysis buffer and colony PCR methods can be used to amplify andsequence the 16S rDNA gene (Example 18). Alternatively, colonies may bestreaked to purity in several passages on solid media. Well separatedcolonies are streaked onto the fresh plates of the same kind andincubated for 48-72 hours at 37° C. The process is repeated multipletimes in order to ensure purity. Pure cultures can be analyzed byphenotypic- or sequence-based methods, including 16S rDNA amplificationand sequencing as described in Example 18. Sequence characterization ofpure isolates or mixed communities e.g. plate scrapes and sporefractions can also include whole genome shotgun sequencing. The latteris valuable to determine the presence of genes associated withsporulation, antibiotic resistance, pathogenicity, and virulence.Colonies can also be scraped from plates en masse and sequenced using amassively parallel sequencing method as described in Example 7. suchthat individual 16S signatures can be identified in a complex mixture.Optionally, the sample can be sequenced prior to germination (ifappropriate DNA isolation procedures are used to lsye and release theDNA from spores) in order to compare the diversity of germinable specieswith the total number of species in a spore sample. As an alternative orcomplementary approach to 16S analysis, MALDI-TOF-mass spec can also beused for species identification (Barreau M, Pagnier I, La Scola B. 2013.Improving the identification of anaerobes in the clinical microbiologylaboratory through MALDI-TOF mass spectrometry. Anaerobe 22: 123-125).

Example 21. Microbiological Strain Identification Approaches

Pure bacterial isolates can be identified using microbiological methodsas described in Wadsworth-KTL Anaerobic Microbiology Manual(Jouseimies-Somer H, Summanen P H, Citron D, Baron E, Wexler H M,Finegold S M. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual), andThe Manual of Clinical Microbiology (ASM Press, 10th Edition). Thesemethods rely on phenotypes of strains and include Gram-staining toconfirm Gram positive or negative staining behavior of the cellenvelope, observance of colony morphologies on solid media, motility,cell morphology observed microscopically at 60× or 100× magnificationincluding the presence of bacterial endospores and flagella. Biochemicaltests that discriminate between genera and species are performed usingappropriate selective and differential agars and/or commerciallyavailable kits for identification of Gram negative and Gram positivebacteria and yeast, for example, RapID tests (Remel) or API tests(bioMerieux). Similar identification tests can also be performed usinginstrumentation such as the Vitek 2 system (bioMerieux). Phenotypictests that discriminate between genera and species and strains (forexample the ability to use various carbon and nitrogen sources) can alsobe performed using growth and metabolic activity detection methods, forexample the Biolog Microbial identification microplates. The profile ofshort chain fatty acid production during fermentation of particularcarbon sources can also be used as a way to discriminate between species(Wadsworth-KTL Anaerobic Microbiology Manual, Jousimies-Somer, et al2002). MALDI-TOF-mass spectrometry can also be used for speciesidentification (as reviewed in Anaerobe 22:123).

Example 22. Construction of an In Vitro Assay to Screen for Combinationsof Microbes Inhibitory to the Growth of Pathogenic E. coli

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of E. coli by modifying the media used forgrowth of the pathogen inoculum. One of several choices of media is usedfor growth of the pathogen such as Reinforced Clostridial Media (RCM),Brain Heart Infusion Broth (BHI) or Luria Bertani Broth (LB) (also knownas Lysogeny Broth). E. coli is quantified by using alternative selectivemedia specific for E. coli or using qPCR probes specific for thepathogen. For example, aerobic growth on MacConkey lactose mediumselects for enteric Gram negatives, including E. coli. qPCR is conductedusing probes specific for the shiga toxin of pathogenic E. coli.

Example 23. Construction of an In Vitro Assay to Screen for Combinationsof Microbes Inhibitory to the Growth of Vancomycin-ResistantEnterococcus (VRE)

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of Vancomycin-Resistant Enterococcus spp. (VRE)by modifying the media used for growth of the pathogen inoculum. Severalchoices of media are used for growth of the pathogen such as ReinforcedClostridial Media (RCM), Brain Heart Infusion Broth (BHI) or LuriaBertani Broth (LB). VRE is quantified by using alternative selectivemedia specific for VRE or using qPCR probes specific for the pathogen.For example, m-Enterococcus agar containing sodium azide is selectivefor Enterococcus spp. and a small number of other species. Probesspecific to the van genes conferring vancomycin resistance are used inthe qPCR.

Example 24. Testing of Bacterial Composition Against Salmonella

The in vitro assay is used to screen for combinations of bacteriainhibitory to the growth of Salmonella spp. by modifying the media usedfor growth of the pathogen inoculum. Several choices of media are usedfor growth of the pathogen such as Reinforced Clostridial Media (RCM),Brain Heart Infusion Broth (BHI) or Luria Bertani Broth (LB). Salmonellaspp. are quantified by using alternative selective media specific forSalmonella spp. or using qPCR probes specific for the pathogen. Forexample, MacConkey agar is used to select for Salmonella spp. and theinvA gene is targeted with qPCR probes; this gene encodes an invasionprotein carried by many pathogenic Salmonella spp. and is used ininvading eukaryotic cells.

Example 25. Method of Preparing the Bacterial Composition forAdministration to a Subject

Two or more strains that comprise the bacterial composition areindependently cultured and mixed together before administration. Bothstrains are independently be grown at 37° C., pH 7, in a GMM or otheranimal-products-free medium, pre-reduced with 1 g/L cysteine HCl. Aftereach strain reaches a sufficient biomass, it is preserved for banking byadding 15% glycerol and then frozen at −80° C. in 1 ml cryotubes.

Each strain is then be cultivated to a concentration of 10¹⁰ CFU/mL,then concentrated 20-fold by tangential flow microfiltration; the spentmedium is exchanged by diafiltering with a preservative mediumconsisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphatebuffer, or other suitable preservative medium. The suspension isfreeze-dried to a powder and titrated.

After drying, the powder is blended with microcrystalline cellulose andmagnesium stearate and formulated into a 250 mg gelatin capsulecontaining 10 mg of lyophilized powder (10⁸ to 10¹¹ bacteria), 160 mgmicrocrystalline cellulose. 77.5 mg gelatin, and 2.5 mg magnesiumstearate.

A bacterial composition can be derived by selectively fractionating thedesired bacterial OTUs from a raw material such as but not limited tostool. As example we prepared a 10% w/v suspension of human stoolmaterial in PBS that is filtered, centrifuged at low speed, and then thesupernate containing spores is mixed with absolute ethanol in a 1:1ratio and vortexed to mix. The suspension is incubated at roomtemperature for 1 hour. After incubation the suspension is centrifugedat high speed to concentrate spores into a pellet containing a purifiedspore-containing preparation. The supernate is discarded and the pelletresuspended in an equal mass of glycerol, and the purified sporepreparation is placed into capsules and stored at −80 degrees Celsius;this preparation is referred to as an ethanol-treated spore population.

Example 26. Method of Treating a Subject with a Bacterial Composition

A subject has suffered from recurrent bouts of C. difficile. In the mostrecent acute phase of illness, the subject is treated with an antibioticsufficient to ameliorate the symptoms of the illness. In order toprevent another relapse of C. difficile, the subject is administered oneof the present bacterial compositions. Specifically, the subject isadministered one of the present bacterial compositions at a dose in therange of 1e10⁷ to 1e10¹² in a lyophilized form, in a gelatin capsulecontaining 10 mg of lyophilized bacteria and stabilizing components. Thesubject takes the capsule by mouth and resumes a normal diet after 4, 8,12, or 24 hours. In another embodiment, the subject may take the capsuleby mouth before, during, or immediately after a meal. In a furtherembodiment, the subject takes the dose daily for a specified period oftime.

Stool is collected before and after treatment. In one embodiment stoolis collected at 1 day, 3 days, 1 week, and 1 month after administration.The presence of C. difficile is found in the stool before administrationof the bacterial composition, but stool collections after administrationshow reducing (such as at least 50% less, 60%, 70%, 80%, 90%, or 95%) tono detectable levels of C. difficile, as measured by qPCR, as describedabove. ELISA for toxin protein or traditional microbiologicalidentification techniques may also be used.

As another measure of subject success, a positive response may bedefined as absence of diarrhea, which itself is defined as 3 or moreloose or watery stools per day for at least 2 consecutive days or 8 ormore loose or watery stools in 48 hours, or persisting diarrhea (due toother causes) with repeating (three times) negative stool tests fortoxins of C. difficile.

Treatment failure is defined as persisting diarrhea with a positive C.difficile toxin stool test or no reduction in levels of C. difficile, asmeasured by qPCR sequencing. ELISA or traditional microbiologicalidentification techniques may also be used.

Example 27. Microbiological Strain Identification Approaches

Pure bacterial isolates are identified using microbiological methods asdescribed in Wadsworth-KTL Anaerobic Microbiology Manual(Jouseimies-Somer H, Summanen P H, Citron D, Baron E, Wexler H M,Finegold S M. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual), andThe Manual of Clinical Microbiology (ASM Press, 10th Edition). Thesemethods rely on phenotypes of strains and include Gram-staining toconfirm Gram positive or negative staining behavior of the cellenvelope, observance of colony morphologies on solid media, motility,cell morphology observed microscopically at 60× or 100× magnificationincluding the presence of bacterial endospores and flagella. Biochemicaltests that discriminate between genera and species are performed usingappropriate selective and differential agars and/or commerciallyavailable kits for identification of Gram negative and Gram positivebacteria and yeast, for example, RapID tests (Remel) or API tests(bioMerieux). Similar identification tests can also be performed usinginstrumentation such as the Vitek 2 system (bioMerieux). Phenotypictests that discriminate between genera and species and strains (forexample the ability to use various carbon and nitrogen sources) can alsobe performed using growth and metabolic activity detection methods, forexample the Biolog Microbial identification microplates. The profile ofshort chain fatty acid production during fermentation of particularcarbon sources can also be used as a way to discriminate between species(Wadsworth-KTL Anaerobic Microbiology Manual, Jousimies-Somer, et al2002). MALDI-TOF-mass spectrometry can also be used for speciesidentification (as reviewed in Anaerobe 22:123).

Example 28. Computational Prediction of Network Ecologies

Source data comprising a genomic-based characterization of a microbiomeof individual samples are used as input computationally delineatenetwork ecologies that would have biological properties that arecharacteristic of a state of health and could catalyze a shift from astate of microbial dysbiosis to a state of health. Applicants obtained16S and metagenomic sequence datasets from public data repositories (seee.g. The Human Microbiome Project Consortium. 2012. Structure, functionand diversity of the healthy human microbiome. Nature 486: 207-214. Dataaccessible at URL: hmpdacc.org) and MetaHit Project (Arumugam M, Raes J,Pelletier E, Paslier D L, Yamada T, Mende D R, Fernandes G R, Tap J,Bruls T, Batto J-M, et al. 2011. Enterotypes of the human gutmicrobiome. Nature 473: 174-180. Data accessible at URL: metahit.eu) forrelevant microbiome studies in multiple disease indications includingCDAD, Type 2 Diabetes, Ulcerative Colitis, and Irritable Bowel Disease,or generated data sets from samples directly using the methods describedin Examples 18 and 19 and further described in the literature (see e.g.Aagaard K, Riehle K, Ma J, Segata N, Mistretta T-A, Coarfa C, Raza S,Rosenbaum S, Van den Veyver I, Milosavljevic A, et al. 2012. AMetagenomic Approach to Characterization of the Vaginal MicrobiomeSignature in Pregnancy ed. A. J. Ratner. PLoS ONE 7: e36466. JumpstartConsortium Human Microbiome Project Data Generation Working Group. 2012.Evaluation of 16S rDNA-Based Community Profiling for Human MicrobiomeResearch ed. J. Ravel. PLoS ONE 7: e39315. The Human Microbiome ProjectConsortium. 2012. Structure, function and diversity of the healthy humanmicrobiome. Nature 486: 207-214.). Nucleic acid sequences are analyzedand taxonomic and phylogenetic assignments of specific OTUs are madeusing sequence similarity and phylogenetic methods that are well knownto those skilled in the art, including but not limited to maximumlikelihood phylogenetic reconstruction (see e.g. Liu K, Linder C R, andWarnow T. 2011. RA×ML and FastTree: Comparing Two Methods forLarge-Scale Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731.McGuire G, Denham M C, and Balding D J. 2001. Models of sequenceevolution for DNA sequences containing gaps. Mol. Biol. Evol 18:481-490. Wróbel B. 2008. Statistical measures of uncertainty forbranches in phylogenetic trees inferred from molecular sequences byusing model-based methods. J. Appl. Genet. 49: 49-67.) From thesetaxonomic assignments OTUs and clades in the dataset are defined usingthe method described in Examples 18 and 19. The certainty of the OTUcall is defined based on the OTU's sequence similarity to a referencenucleic acid sequence and the proximity of the OTU sequence relative toone or more reference sequences in the phylogeny. The specificity of anOTU's taxonomic and phlylogenetic assignment determines whether thematch is assigned at the level of Family, Genus, Species, or Strain, andthe confidence of this assignment is determined based on the position ofbootstrap supported branches in the reference phylogenetic tree relativeto the placement of the OTU sequence being interrogated. In addition,microbial OTU assignments may be obtained from assignments made inpeer-reviewed publications.

Applicants designated individual subject samples to biologicallyrelevant sample phenotypes such as but not limited to “healthy state,”“recurrent Clostridium difficile infection,” “Crohn's disease,” “InsulinResistance,” “Obesity,” “Type 2 diabetes,” “Ulcerative Colitis”. In oneembodiment samples are assigned to “health” and “disease” phenotypes. Inanother embodiment, samples are assigned higher resolution phenotypesuch as but not limited to: “health:human”, “health:mouse”,“health:human microbiome project”, “health:microbiota donor”,“health:microbiota recipient”, “disease:microbiota recipient”, or“disease:no treatment”, “disease:human”, or “disease:mouse”. In anotherembodiment, samples where assigned to higher resolution phenotypes, suchas but not limited to those defined that characterize phenotypesspecific to samples from fecal donors and patients who received a fecalmicrobial transplant from these donors.

In another embodiment, other phenotypes that define a category ofdisease or health that represents the underlying state of the populationunder study can be used. Applicants then computationally determined themicrobial network ecologies for each phenotype using the OTU and cladeassignments that comprise the microbial profile for each sample and thealgorithms described above in the Section entitled “Method ofDetermining Network Ecologies.”

Importantly, Network Ecologies that represent a state of health in onedisease indication can represent states of health in additional diseasestates. Additionally, Keystone OTUs found in a network associated withhealth for different disease indications can overlap. Applicants foundthat a large number of network ecologies overlapped particularly betweenthose associated with health in the cases of CDAD and Type 2 Diabetesdespite the analysis of substantially different genomic data sets forthe two diseases.

Example 29. Identification of Network Classes, Keystone OTUs, Clades,and Functional Modalities Identification of Keystone OTUs, Clades andFunctions

The human body is an ecosystem in which the microbiota and themicrobiome play a significant role in the basic healthy function ofhuman systems (e.g. metabolic, immunological, and neurological). Themicrobiota and resulting microbiome comprise an ecology ofmicroorganisms that co-exist within single subjects interacting with oneanother and their host (i.e., the mammalian subject) to form a dynamicunit with inherent biodiversity and functional characteristics. Withinthese networks of interacting microbes (i.e. ecologies), particularmembers can contribute more significantly than others; as such thesemembers are also found in many different ecologies, and the loss ofthese microbes from the ecology can have a significant impact on thefunctional capabilities of the specific ecology. Robert Paine coined theconcept “Keystone Species” in 1969 (see Paine R T. 1969. A note ontrophic complexity and community stability. The American Naturalist 103:91-93) to describe the existence of such lynchpin species that areintegral to a given ecosystem regardless of their abundance in theecological community. Paine originally describe the role of the starfishPisaster ochraceus in marine systems and since the concept has beenexperimentally validated in numerous ecosystems.

Keystone OTUs, Phylogenetic Clades (a.k.a. Clades), and/or Functions(for example, but not limited to, KEGG Orthology Pathways) arecomputationally-derived by analysis of network ecologies elucidated froma defined set of samples that share a specific phenotype. Keystone OTUs,Clades and/or Functions are defined as all Nodes within a defined set ofnetworks that meet two or more of the following criteria. UsingCriterion 1, the node is frequently observed in networks, and thenetworks in which the node is observed are found in a large number ofindividual subjects; the frequency of occurrence of these Nodes innetworks and the pervasiveness of the networks in individuals indicatesthese Nodes perform an important biological function in manyindividuals. Using Criterion 2, the node is frequently observed innetworks, and the Node is observed contains a large number of edgesconnecting it to other nodes in the network. These Nodes are thus“super-connectors”, meaning that they form a nucleus of a majority ofnetworks and as such have high biological significance with respect totheir functional contributions to a given ecology.

In another embodiment a Keystone Node is defined as one that occurs in asample phenotype of interest such as but not limited to “health” andsimultaneously does not occur in a sample phenotype that is not ofinterest such as but not limited to “disease.” Optionally, a KeystoneNode is defined as one that is shown to be significantly different fromwhat is observed using permuted test datasets to measure significance.In another embodiment of Criterion 2 Keystone OTUs, Clades, or Functionscan be defined using a hierarchical clustering method that clustersNetworks based on their OTU, Clade, or functional pathways.Statistically significant branch points in the hierarchy are definedbased on the topological overlap measure; this measure is a highlyrobust measure of network interconnectedness (Langfelder P, Zhang B,Horvath S. 2008. Defining clusters from a hierarchical cluster tree: theDynamic Tree Cut package for R. Bioinformatics 24: 719-720.). Once thesebranch points are defined the Keystones are delineated as OTUs, cladesor functional pathways that are found consistently across all networksin all or a subset of the network clusters.

Importantly, we identify the absence of Keystone OTUs in multipleparticular disease states, indicating that bacterial compositionscomprised of specific sets of Keystone OTUs are likely to have utilityin multiple disease indications.

Example 30. Network Analysis Across Multiple Data Sets and Selection ofTarget Network Ecologies with Capacity to Sporulate

One can select Network Ecologies and/or Network Class Ecologies as leadtargets by defining networks with a specific biological function oractivity such as sporulation. Networks Ecologies or Network ClassEcologies are first selected as described above. In one example, allNetwork Ecologies or Network Class Ecologies that contain at least oneOTU that is capable of forming spores are targeted. In another example,all Network Ecologies or Network Class Ecologies that contain at leastone OTU that is capable of forming spores, and that are comprised of atleast 50%, 75%, or 100% Keystone OTUs are targeted. Keystone OTUs areselected as described above. OTUs are defined as spore formers usingeither phenotypic assays (see e.g. Stackebrandt and Hippe. Taxonomy andSystematics. In Clostridia. Biotechnology and Medical Applications.) orgenetic assays (see e.g. Abecasis A B, Serrano M, Alves R, Quintais L,Pereira-Leal J B, and Henriques A O. 2013. A genomic signature and theidentification of new sporulation genes. J. Bacteriol.; Paredes-Sabja D,Setlow P, and Sarker M R. 2011. Germination of spores of Bacillales andClostridiales species: mechanisms and proteins involved. TrendsMicrobiol. 19: 85-94).

Clade membership of bacterial OTUs is based on 16S sequence data. Cladesare defined based on the topology of a phylogenetic tree that isconstructed from full-length 16S sequences using maximum likelihoodmethods familiar to individuals with ordinary skill in the art ofphylogenetics. Clades are constructed to ensure that all OTUs in a givenclade are: (i) within a specified number of bootstrap supported nodesfrom one another, and (ii) within 5% genetic similarity. OTUs that arewithin the same clade can be distinguished as genetically andphylogenetically distinct from OTUs in a different clade based on 16S-V4sequence data, while OTUs falling within the same clade are closelyrelated. OTUs falling within the same clade are evolutionarily closelyrelated and may or may not be distinguishable from one another using16S-V4 sequence data. Members of the same clade, due to theirevolutionary relatedness, play similar functional roles in a microbialecology such as that found in the human gut. Compositions substitutingone species with another from the same clade are likely to haveconserved ecological function and therefore are useful in the presentinvention. All OTUs are denoted as to their putative capacity to formspores and whether they are a Pathogen or Pathobiont (see Definitionsfor description of “Pathobiont”). NIAID Priority Pathogens are denotedas ‘Category-A’, ‘Category-B’, or ‘Category-C’, and OpportunisticPathogens are denoted as ‘OP’. OTUs that are not pathogenic or for whichtheir ability to exist as a pathogen is unknown are denoted as ‘N’. The‘SEQ ID Number’ denotes the identifier of the OTU in the SequenceListing File and ‘Public DB Accession’ denotes the identifier of the OTUin a public sequence repository. For SEQ ID NOs referenced in Table 1,reference is made to e.g., WO2014/121304, which is incorporated byreference herein in its entirety.

Example 31: Selection of Patients and Method for Specimen Collection

Paired stool samples and blood specimens are collected and stored weeklyover the course of the transplant hospitalization including prior toconditioning, as well as on days 0, 7, 14, 21, 30, 60, and 100. ForChronic GVHD, samples are collected post GVHD at day 105, day 120, day180.

Skin, lung, vaginal and oral samples are obtained as well pre- andpost-transplant. Intestinal biopsies samples are saved from patientssubjected to such analysis. Stool samples from patients are stored at 4°C. for <24 h before freezing at −80° C. GVHD is diagnosed clinically,confirmed pathologically by biopsy whenever possible, and classifiedaccording to standard criteria. Patients are evaluated for acute GVHDbased on historical consensus criteria as described previously (seeRowlings P A, Przepiorka D, Klein J P, et al. IBMTR Severity Index forgrading acute graft-versus-host disease: retrospective comparison withGlucksberg grade. Br J Haematol. 1997). Cases of GVHD are furthercategorized by treatment with or without systemic steroids (prednisoneor methylprednisolone, 0.5 mg/kg daily or higher). Cause of death isdetermined using a standard algorithm where outcomes are prioritized inthe following order: 1) primary disease recurrence, 2) graft failure, 3)GVHD, 4) infection, and 5) organ failure. Thus in patients withoutdisease recurrence or graft failure, those who are being treated forGVHD at the time of death are considered to have succumbed toGVHD-related mortality, including those who died from infections.

Example 32. Cross Niche Analysis of Microbiome

DNA is extracted from samples from various sites—gut, blood, lung,vaginal, oral and skin. Extracted DNA is subjected to 16S, ITS or 18Ssequencing as described elsewhere. Nucleic acid sequences are analyzedto define taxonomic assignments using sequence similarity andphylogenetic placement methods or a combination of the two strategies.These methods map a sequence read to a reference database and selectingthe match with the best score and e-value. Common databases include, butare not limited to the Human Microbiome Project, NCBI non-redundantdatabase, Greengenes, RDP, and Silva for taxonomic assignments.Microbial clades are assigned using databases including but not limitedto MetaPhlAn. Phylogenetic abundance comparisons are performed usinglinear discriminant analysis (LDA) effect size (LEfSe) analysis, using alogarithmic LDA cutoff of 2.0. Taxonomic and phylogenetic abundance datafrom various niches is then subjected to comparative analysis toidentify microbes that are unique to each site vs those that overlapbetween two or more sites.

Example 33. Selection of Microbes to be Used for Mitigating GVHD andOther Immune Based Diseases Based on Microbiome Analysis

Candidate microbes to be used for mitigating GVHD and other immune baseddisorder are selected based on microbiome analysis of samples fromsingle or multiple niche. For example, microbes that are highly abundantin patients who do not succumb to GVHD or are alive post bone marrowtransplantation are associated with low or no GVHD incidence andsurvival post GVHD. These microbes could be abundant at a single sitesuch as the gut. Alternatively, these microbes are also abundant atanother site in addition such as the skin, lung, vagina and oral or atall of these sites within a diseased subject. Alternatively, microbesare selected based on their abundance at one site such as the gut andnot at other sites. These microbes are then tested in in vitro and invivo models to test their ability to inhibit inappropriate immuneresponses and reduce inflammation at multiple disease target sites suchas but not limited to gut, liver, kidney, lung and skin.

Example 34. Selection of a Metabolically Altered Organism Based onPrebiotic Fermentation

Bacterial isolates or evolved laboratory strains are inoculated in 25 mlof Versa TREK REDOX 2 broth (Trek Diagnostic Systems) supplemented with30% sterile-filtered cow rumen fluid and prebiotic of interest incubatedunder anaerobic conditions for 2 days at 37° C. Following incubation,cultures are centrifuged at 6000 rpm for 10 min. Supernatants,uninoculated medium, or standards of short chain fatty acids (e.g.,acetate, propionate, butyrate (Sigma-Aldrich) are injected into a PerkinElmer Autosystem XL Gas Chromatograph containing a Supelco packed column(Sigma-Aldrich) according to the manufacturer's protocol (Dairy OneCooperative) (Foditsch C et al., 2014. Isolation and Characterization ofFaecalibacterium prausnitzii from Calves and Piglets. PLoS One. 9(12):e116455). Bacterial isolates are selected with maximal short chain fattyacid (e.g., butyrate) production. Similar kind of analysis is done toaccess impact of prebiotics on other bacterial metabolite productionsuch as but not limited to secondary bile acids.

Example 35. Mouse Model to Study the Impact of Microbiome in Acute GraftVersus Host Disease

A number of experimental models for studying acute GVHD exist andinvolve the transplantation of T-cell-depleted bone marrow supplementedwith varying numbers and phenotypic classes of donor lymphocytes (eithersplenocytes or lymph node T cells) into lethally irradiated recipients.The severity of aGvHD depends on several factors—1) the dose and type ofT-cell subsets (i.e. CD4+, CD8+ or TReg cells), 2) Irradiation dose, 3)Genetic disparities (MHC, miHAs), 4) Variation in environmentalpathogens between labs and in mice from different suppliers. AllogeneicGVHD mouse models can be MHC-mismatched and miHA-mismatched. The morerecently developed xenogeneic GVHD mouse models involve transplantationof human cells into immunodeficient mice. Both these models areextensively in Schroeder and DiPersio, 2011 Disease Models & Mechanisms.

To study the impact of microbiome on acute GVHD using the allogeneicGVHD mouse models, recipient mice such as BALB/c (H2d) or C57BL/6 (H2b)mice are treated with a gut-decontaminating antibiotic cocktail(ampicillin and vancomycin) to mimic microbiota injury that occurs inallo BMT patients. Mice are then exposed to a myeloablative dose oftotal body irradiation (TBI, 11 Gy) and then transplanted by intravenousinjection with bone marrow and purified T cells from fullyMHC-mismatched C57/Bl6 (H2b) or B10.BR mice respectively. Alternatively,xenogeneic GVHD mouse models are utilized where the immunodeficientNOD.scid (IL-2Rγc)−/− (NOG) or NOD.scid (Il2rgmut) (NSG) mice aretreated with a gut-decontaminating antibiotic cocktail (ampicillin andvancomycin), transplanted with human PBMCs by I.V. or I.P. injection andsubsequently exposed to a myeloablative dose of total body irradiation(2.5 Gy).

Example 36. Murine Model to Study the Impact of Microbiome in ChronicGraft Versus Host Disease

Current mouse models of cGvHD (Schroeder and DiPersio, 2011) can bebroadly divided into sclerodermatous (pro-fibrotic) models,autoantibody-mediated (lupus-like) models and a more recently reportedmodel in which thymic function is defective (Sakoda et al., 2007; Chuand Gress, 2008). Herein is an example with a validated sclerodermatous(pro-fibrotic) models model. Recipient mice BALB/c (H2d) or C57/Bl6(H2b) are treated with a gut-decontaminating antibiotic cocktail(ampicillin and vancomycin) to mimic microbiota injury that occurs inallo BMT patients. Mice are then exposed to a myeloablative dose oftotal body irradiation (700-900 cGy ore 900-1100 cGy respectively) andthen transplanted by intravenous injection with bone marrow and purifiedT cells or splenocytes from B10.D2 (H2c) or LP/J (H2b) micerespectively.

Example 37. Culturing and Banking Bacterial Isolates from Mouse or HumanFeces

Entire stool specimens are collected and homogenized in 1-3 volumes of0.05% peptone using a sterile stainless steel blender with 1-3 volumesof peptone. Approximately 1 gram of the specimen is serially diluted(10-fold) in pre-reduced, anaerobically sterilized (PRAS) dilutionblanks (Anaerobe Systems). A separate ˜1 gram aliquot is weight, driedin a vacuum over, and re-weighed in order to calculate counts on adry-weight basis. To select for Clostridiales bacteria, includingBlautia species, 100 μL of the homogenized stool sample dilution seriesis plated on Brain-Heart Infusion blood agar (SBA, Becton Dickinson)supplemented with 4 μg/mL trimethoprim (Sigma Chemical) and 1 μg/mLsulfamethoxazole (Sigma), Brucella Blood Agar (BAP, Anaeobe Systems),CDC ANA blood agar, (BBL Microbiology Systems), and egg yolk agar (EYA,Anaerobe Systems) (Finegold S M, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe E M, Collins M D,Lawson P A, Summanen P, Baysallar M, Tomzynski T J, Read E, Johnson E,Rolfe R, Nasir P, Shah H, Haake D A, Manning P, Kaul A, 2002.Gastrointestinal microflora studies in late-onset autism. Clin InfectDis 1:35). To select for spore-formers, the dilutions may be heated at70-80° C. for 10-20 minutes and plated in the same manner as thenon-heated homogenized stool samples. After 5 days of growth at 37° C.in an anaerobic chamber, single colonies are selected. The colonypurification process is repeated by restreaking select single colonies,growing as described above, and selecting again for single colonies.Single colonies are frozen in 15%-25% glycerol in 1 mL cryotubes andstored at −80° C.

Example 38. Administration of Bacterial Isolates, with or withoutPrebiotics, to Mitigate Experimental Acute GVHD

BALB/c (H2d) or C57BL/6 mice in the case of allogeneic models andNOD.scid (IL-2Rγc)−/− (NOG) or NOD.scid (Il2rgmut) (NSG) in the case ofxenogeneic models are treated with oral vancomycin and ampicillin.Following decontamination, mice are housed in autoclaved conditions(caging, bedding, water and food) to eliminate nearly all endogenousbacteria present within the flora of mice. Mice are then treated bygavage with a liquid suspension of the cultured bacterial isolate,optionally with one or more prebiotic carbohydrates. Mice are thenexposed to a myeloablative dose of total body irradiation and thentransplanted by intravenous injection with bone marrow and purified Tcells from fully MHC-mismatched C57/Bl6 (H2b) or B10.BR mice forallogeneic models and human PBMCs for xenogeneic models. Effect on organpathology, weight and overall survival is measured as described.

Example 39. Administration of Bacterial Isolates, with or withoutPrebiotics, to Mitigate Experimental Chronic GVHD

Recipient mice BALB/c (H2d) or C57/Bl6 (H2b) are treated with agut-decontaminating antibiotic cocktail (ampicillin and vancomycin).Following decontamination, mice are housed in autoclaved conditions(caging, bedding, water and food) to eliminate nearly all endogenousbacteria present within the flora of mice. Mice are then treated bygavage with a liquid suspension of the cultured bacterial isolate inaddition to one or more prebiotic carbohydrates. Mice are then exposedto a myeloablative dose of total body irradiation and then transplantedby intravenous injection with bone marrow and purified T cells orsplenocytes from B10.D2 (H2c) or LP/J (H2b) mice respectively. Mice areevaluated for fibrotic changes in the dermis, which can involve thelung, liver and salivary glands beginning day 30 post transplantation.

Example 40. GVHD Clinical and Histological Scoring

Mice are monitored daily for survival and weekly for GVHD clinicalscores (see Cooke, K. R., L. Kobzik, T. R. Martin, J. Brewer, J.Delmonte Jr., J. M. Crawford, and J. L. Ferrara. 1996. An experimentalmodel of idiopathic pneumonia syndrome after bone marrowtransplantation: I. The roles of minor H antigens and endotoxin. Blood.88:3230-3239). Small intestine, large intestine, and liver samples areevaluated histologically for evidence of GVHD and scored as previouslydescribed (see Hill, G. R., J. M. Crawford, K. R. Cooke, Y. S. Brinson,L. Pan, and J. L. Ferrara. 1997. Total body irradiation and acutegraft-versus-host disease:the role of gastrointestinal damage andinflammatory cytokines. Blood. 90:3204-3213).

Example 41. Measuring Paneth Cell Numbers and Functionality

The small intestinal lumens of adult mice are rinsed with ice-cold waterand segmented. Crypts are eluted by first turning the segments insideout and then shaking them in PBS containing 30 mM EDTA and lacking Ca²⁺and Mg²⁺. The eluted villi and crypts are pelleted at 700×g, resuspendedin PBS, and transferred to siliconized microfuge tubes using capillarypipettes. The crypts are resuspended in iPIPES buffer (10 mM PIPES (pH7.4) and 137 mM NaCl) in preparation of exposure to secretory stimuli.

Crypts are incubated in 30 μl of iPIPES containing 1000 bacterial(Clostridiales) CFU per crypt for 30 min at 37° C. Cellular componentsare pelleted by brief centrifugation, and supernatants transferred tosterile microfuge tubes and stored at −20° C. This method may be scaledup using up to ˜3000 crypts in 2 ml iPIPES (plus or minus Clostridialesbacteria). Crypts are pelleted and 10 μL of the supernatants areanalyzed for bactericidal activity against Clostriales and Enterococcusbacteria in liquid culture or on agar plates. Proteins are extractedfrom the rest of the supernatant as well as the crypts using 30% aceticacid. Total protein extracted from each fraction was resolved by AU-PAGEand subjected to western blot analysis using anti-cryptdin-1 as follows.Proteins from AU-PAGE are transferred to a nitrocellulose membrane. Themembrane is then blocked with 5% skim milk, incubated sequentially withanti-rabbit mouse cryptdin-1 (1:500), horseradish peroxidase-conjugatedanti-rabbit IgG (1:20,000) and chemiluminescent substrate (SuperSignal,Pierce, Rockland, Ill.), and visualized (Ayabe T, Satchell D P, Wilson CL, Parks W C, Selsted M E, Ouellette A J, 2000. Secretion ofmicrobicidal α-defensins by intestinal Paneth cells in response tobacteria. Nature Immunology 1:113-118).

Example 42. Measuring Intestinal Crypt Regeneration by Organoid Growth

Organoid formation may be used as a proxy for intestinal cryptregeneration as follows:

Lgr5-EGFP-IRES-CreERT2 knock-in (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, JAXmice #008875) and ROSA26-tdTomato(B6.Cg-Gt(ROSA)26Sortml4(CAG-tdTomato)Hze/J, JAX mice #007914) mice arepurchased from the Jackson Laboratory (Bar Harbor, Me., USA) and crossedto produce Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice (LRT mice). Theobtain persistent labeling of Lgr5+ stem cells with tdTomato (as well astdTomato-labeled progeny of Lgr5+ stem cells), the LRT mice areadministered 4-hydroxytamoxifen (4-OHT; Sigma Aldrich)intraperitoneally, once, at 10-20 days old.

To obtain single crypt cells for in vitro analyses, the LRT mice aresacrificed at 3-5 weeks old, and the duodenum and jejunum (10 cm fromthe stomach) are harvested and rinsed three times with coldphosphate-buffered saline (PBS−). The intestinal tubes are openedlongitudinally and the villi are scraped using a coverslip, then washedthree times in cold 1×PBS−. The intestinal tubes are cut into 2-3 mmpieces and suspended and extensively washed in 1×PBS−+2% fetal bovineserum (FBS). Then, the 2-3-mm pieces are treated with 50 mM EDTA/1×PBS−for 30 min at 4° C. on a rocking platform to dissociate the crypts fromthe intestinal tubes. The dissociated crypts are passed through a 70-μmcell strainer, washed once with 1×PBS−, and treated with TrypLE Express(Life Technologies, Carlsbad, Calif., USA) for 30 min at 37° C. Then,the dissociated cells are passed through a 40-μm strainer andsubsequently, through a 20-μm strainer. The strained cells are pelleted,resuspended in 1×PBS−+2% FBS, and used as single crypt cells.

The isolated single crypt cells are cultured in organoid medium[advanced DMEM/F12 supplemented with 1× GlutaMAX, 10 mM HEPES, 1×penicillin/streptomycin, 1×N2, 1×B27 (all obtained from LifeTechnologies, Carlsbad, Calif., USA), N-acetylcysteine (Sigma-Aldrich,St Louis, Mo., USA, 1 mM), murine epidermal growth factor (LifeTechnologies, Carlsbad, Calif., USA, 50 ng/ml), murine Noggin(Peprotech, Rocky Hill, N.J., USA, 100 ng/ml), and murine R-Spondin I(R&D Systems, Minneapolis, Minn., USA, 1 μg/ml)]. During the first twodays of culturing 10 μM of Rho kinase inhibitor Y-27632 (Sigma-Aldrich,St Louis, Mo., USA) is added.

350 μM of organoid medium is added into wells of 48-well plates, andthen single crypt cells are plated to the wells (1×105 cells/10 μl1×PBS−+2% FBS per well). Matrigel (BD, Franklin Lakes, N.J., USA) isadded to a final concentration of 10%. The organoid medium isreplenished every day for the first 3 days and every 2-3 daysthereafter. The cells are incubated in a humidified CO2 incubator at 37°C. After 12 days, the number of organoids is counted by phase-contrastmicroscopy with a 4× objective (Yamauchi M, Otsuka K, Kondo H, Hamada N,Tomita M, Takahashi M, Nakasono S, Iwasaki T, Yoshida K, 2014. A novelin vitro survival assay of small intestinal stem cells after exposure toionizing radiation. J Radiat Res. 55:381-390).

To investigate organoid growth over time, time-lapse microscopy isperformed using a confocal laser microscope (C1si, Nikon, Japan) andimages are analyzed using ImageJ software (National Institutes ofHealth, Bethesda, Md., USA).

Example 43. Measuring Intestinal Crypt Regeneration by Quantitative RealTime PCR

Alternatively, C57BL/6J mice may be used to study intestinalregeneration using a real time PCR (qPCR) method. Duodenum and jejunumare harvested as described in Example 20 and cut into 1 cm small pieces.The tissue pieces are placed in a FastPrep-24 Lysing Matrix D tubes (MPBiomedicals, Solon, Ohio) filled with 600 μL of RLT lysis buffer(Qiagen, Valencia, Calif.). Next, 6 μL (β-mercaptoethanol(Sigma-Aldrich, St. Louis, Mo.) is added to the lysing tubes. And totalRNA is isolated from the lysed samples using the Qiagen RNeasy Mini Kitaccording to the manufacturer's instructions. Complimentary DNA is madeusing a High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. qPCR is performed on cDNA samples in triplicate using anApplied Biosystems StepOne Plus system. Individual probes forLgr5(Mm00438890_m1), Ascl2 (Mm01268891_g1), Bmi1(Mm03053308_g1), Olfm4(Mm01320260_m1), and mTert(Mm01352136_m1) from Applied Biosystems areused and data analyzed using a relative standard curve method normalizedto β-actin(Mm00607939_s1; Applied Biosystems). Actively cyclingintestinal stem cells are labeled by Lgr5, Ascl2, and Olfm4, whereasslowly cycling (quiescent) intestinal stem cells are labeled by mTertand Bmi1 (Dehmer J J, Garrison A P, Speck K E, Dekaney C M, VanLandeghem L, Sun X, Henning S J, Helmrath M A, 2011. Expansion ofintestinal epithelial stem cells during murine development. PLoS One.6:e27070).

Example 44. Measurement of Transepithelial Electrical Resistance

The following protocol is preferentially used for monocultures ofintestinal epithelial cells but may also be applied to epithelial cellsderived from other organs such as the vagina or liver. Monolayers ofepithelial cells (e.g., Caco-2) are obtained from the ATCC or frompatient biopsy and maintained in Dulbeco's Modified Eagle Medium with10% fetal bovine serum or RPMI 1640 with 10% fetal bovine serum.

A monolayer is formed by seeding epithelial cells grown to 80-90%confluency (˜10̂5 cells/cm2) on transwell plates (Corning) and incubatingthe plates between at 37° C. and 5% CO2. The cells are incubated for 10days, during which they are fed with fresh medium (basolaterally and/orapically) every other day. The integrity of the cell layer is assessedby transepithelial electrical resistance (TEER) using Millicell-ERSequipment (Millipore) and a World Precision Instruments probe (WPIaccording to the manufacturers' instructions.

Example 45. Dye-Based Evaluation of Epithelial Integrity

Cells from ATCC or patient biopsy are treated as in Example 43. Insteadof measuring TEER, the transwell plates are disassembled and each filterwell is treated with 75 μL 100 μg/mL of the non-membrane permeable dyeLucifer Yellow (Sigma) in Hank's Buffered Salt Solution (HBSS) buffer,pH 7.4 (Invitrogen). Next, 250 μL of HBSS buffer, pH 7.4 is added to thebottom wells and the transwell plates are reassembled and then incubatedfor 2 hours with shaking (60 rpm) at room temperature. Lucifer yellowfluorescence is measured using a Cytofluor II fluorometer at anexcitation wavelength of 485 nm and an emission wavelength of 530 nmPermeability is calculated based on the percentage of Lucifer Yellowthat leaked from the apical chamber to the basolateral chamber of thetranswell plates.

Example 46. Method for Measuring Intestinal Permeability Based onCitrulline Production by Enterocytes

Recently, it was shown that citrulline appeared to be particularlyuseful to detect gut damage, as blood concentrations of this amino aciddirectly reflect functioning small intestinal cell mass (Crenn P, VahediK, Lavergne-Slove A, Cynober L, Matuchansky C, Messing B. Plasmacitrulline: a marker of enterocyte mass in villous atrophy-associatedsmall bowel disease. Gastroenterology 2003). Blood is collected inheparin from each patient through the central venous catheter beforestarting therapy and on each Monday, Wednesday and Friday thereafteruntil discharge. Plasma is prepared and stored at −80 1 C for lateranalysis. Citrulline concentrations (mM) are measured by a standardprocedure for determining amino acid concentrations usinghigh-performance liquid chromatography (Shimadzu, Kyoto, Japan) asdescribed in Herbers et al., 2008. Bacteraemia coincides with lowcitrulline concentrations after high-dose melphalan in autologous HSCTrecipients. Bone Marrow Transplant. 42:345-349.

Example 47. Method for Measuring Levels of Microbes in Distal Organs(Liver, Thymus, Lungs, Kidneys)

Liver, thymus, lungs, and kidneys from mice that had receivedtransplants are removed aseptically and homogenized in 200 μL sterilesaline 0.9%. Then, 100 μL is cultured aerobically on blood agar anddeMan-Rogosa-Sharp agar plates (Difco, Detroit, Mich.), blood agarsupplemented for anaerobes, chocolate blood agar, MacConkey agar, andSabouraud agar for 24 hours in room air supplemented with 10% CO2;colony-forming units are counted and numbers adjusted to weight.Alternatively, genomic DNA from the bacteria is extracted and 16S rDNAsequencing is performed as described

Example 48. Method for Measuring Effect of Microbes, with or withoutPrebiotics, on Bacterial Metabolites Such as SCFA Levels

Short-chain fatty acids (SCFA), which are produced by many bacteria as abyproduct of carbohydrate fermentation. SCFA have been found to beimportant modulators of the immune system. They are abundantly producedbacteria from the Class Clostridia. To evaluate the effect ofadministered bacterial composition, optionally with one or moreprebiotics, on SCFA, fecal pellets are collected to quantify SCFAlevels, particularly acetate, propionate, or butyrate. SCFAs, creatines,and hydroxy-SCFAs are quantified by alkalinizing stool samples,obtaining fingerprints of the metabolic composition of the sample using1D 1H NMR on a Bruker Avance-600 MHz Spectrometer, and analyzing withsupervised multivariate statistical methods using Chenomx NMR Suitesoftware.

Example 49. Administration of Bacterial Metabolites Such as SCFA toMitigate GVHD

Sodium acetate (150 mM) is delivered via the drinking water of micebeginning 2 weeks prior to BMT. Mice are then irradiated andtransplanted with continued supplementation of sodium acetate. Mice areeuthanized to evaluate for pathological evidence of GVHD, as well as toquantify and characterize large intestinal Tregs and alloreactiveeffector T cells by surface staining or intracellular staining followedby flow cytometry on days 14 and 28.

Example 50. Extraction and Purification of ImmunomodulatoryOligosaccharides from Plants

Store-bought plants are cut, lyophilized, and ground into powder. Thepowder (˜500 grams) is extracted three times with 2 L of ethanol, andthe concentrated extract was collected, lyophilized, and resuspendedwith 1 L of distilled water at 85° C. The water-soluble portion isprecipitated by four volumes of ethanol at 4° C. to yieldpolysaccharides. Peptides are removed from this sample by treating withPronase (Roche Applied Science). The resulting sample is run over aBio-Gel P-6 gel filtration column (1.5×90 cm) and eluted with distilledwater containing 0.02% sodium azide at a flow rate of 0.5 ml/min. Allchromatographic fractions containing carbohydrates are analyzed by aphenol-sulfuric acid method (e.g., Masuko T, Minami A, lisaki N, MajimaT, Nishimura S-I, Lee Y C, 2005. Carbohydrate analysis by aphenol-sulfuric acid method in microplate format. 339:69-72) andquantitated by measuring the optical density at 490 nm (Tsai C-C, LinC-R, Tsai H-Y, Chen C-J, Li W-T, Yu H-M, Ke Y-Y, Hsieh W-Y, Chang C-Y,Wu C-Y, Chen S-T, Wong C-H, 2013. The Immunologically ActiveOligosaccharides Isolated from Wheatgrass Modulate Monocytes viaToll-like Receptor-2 Signaling. 288:17689-17697).

Example 51. Selection of Oligosaccharides to Augment Gut Microbiome

The ability of bacterial isolates to grow on a panel of simple andcomplex carbohydrates is evaluated using a phenotypic array whosecomposition has been previously described previously (Martens E C, LoweE C, Chiang H, Pudlo N A, Wu M, et al. 2011. Recognition and degradationof plant cell wall polysaccharides by two human gut symbionts. PLoS Biol9: e1001221). Growth measurements are collected in duplicate over thecourse of 3 days at 37° C. under anaerobic conditions. A total of threeindependent experiments are performed for each species tested (n=6growth profiles/substrate/species). Total growth (A_(tot)) is calculatedfrom each growth curve as the difference between the maximum and minimumoptical densities (OD₆₀₀) observed (i.e., A_(max)−A_(min)). Growth ratesare calculated as total growth divided by time(A_(tot)/(t_(max)−t_(min))), where tmax and tmin correspond to thetime-points at which Amax and Amin, respectively, are collected.

Example 52. Administration of Carbohydrates to Mitigate ExperimentalGVHD

Carbohydrates such as xylose are delivered via the drinking water ofmice beginning 2 weeks prior to BMT. Mice are then irradiated andtransplanted with continued supplementation of xylose. Mice areeuthanized to evaluate for pathological evidence of GVHD, as well as toquantify and characterize large intestinal Tregs and alloreactiveeffector T cells by surface staining or intracellular staining followedby flow cytometry on days 14 and 28. This method may be appliedmonosaccharides, disaccharides, oligosaccharides, polysaccharides, andmixtures thereof.

Example 53. Preventing Graft Versus Host Disease in a Subject

To determine efficacy in preventing GVHD, subjects undergoing allogeneichematopoietic stem cell transplantation are selected. GVHD prophylacticregimen is administered on day −1, where day 0 is day of transplantationor day −1 plus day 17. One arm of the study includes the test article,second arm test article plus standard of care and the third arm isstandard of care alone. Standard GVHD prophylaxis consists ofcyclosporine twice a day starting on day −1 with target troughlevels >200 ng/mL in combination with short course of methotrexate (15mg/sqm on day +1 and 10 mg/sqm on days +3 and +6). Most patientstransplanted from unrelated donors received anti-T-cell globulin (ATGFresenius) at a low dose of 5 mg/kg on days −3 to −1. To estimate theprophylactic and therapeutic effect of test article, the patients arecarefully monitored and documented for the presentation of acute andchronic GVHD symptoms, the time of onset, the severity of the symptoms,the responsiveness to treatment, and the occurrence of infections. Stooland blood samples are collected at pretreatment, day 4, day 14, day 28,3 months, and 6 months.

Example 54. Treating Acute Graft Versus Host Disease in a Subject

To determine the efficacy of treating acute GVHD, test article isadministered to a subject with clinical signs of acute GVHD as describedelsewhere. Test article is orally administered daily either alone or inconjunction with standard of care. At the time of test articleadministration, subjects are at least 10 days post allogeneichematopoietic cell transplantation, have GI symptoms consistent withGrade II GVHD, and have endoscopic evidence of GVHD. The diagnosis ofGVHD is confirmed by biopsy of the intestine (esophagus, stomach, smallintestine, or colon) or skin. Stool, blood and other samples arecollected prior to administration of test article day −2 and day +1, day+7 and Day +10. To evaluate therapeutic effect of test article, thepatients are carefully monitored and documented for the presentation ofacute GVHD symptoms, the severity of the symptoms, the responsiveness totreatment, and the occurrence of infections.

Example 55. Treating Chronic Graft Versus Host Disease in a Subject

Chronic GVHD presents anytime starting day 100 post bone marrowtransplantation. Conventional treatment of chronic GVHD requiresprolonged periods of systemic immunosuppressive therapy with potentdrugs such as corticosteroids and cyclosporine. Agents such asmycophenolate mofetil, rapamycin (sirolimus), imatinib and rituximab areused in patients with steroid-refractory chronic GVHD. To determine theefficacy of treating chronic GVHD, test article is administered to asubject with clinical signs of chronic GVHD as described elsewhere. Testarticle is orally administered daily either alone or in conjunctionstandard of care. At the time of test article administration, subjectsare at least 100 days post allogeneic hematopoietic celltransplantation, have symptoms consistent with chronic GVHD.

Example 56. Immunomodulation of Autologous BMT Recipients

Subjects undergoing autologous hematopoietic stem cell transplantationare selected. Test article is administered on day −1, where day 0 is dayof transplantation or day −1 plus day 17. Subjects are monitoredclinical signs of infections, functionality of organs as well as successof graft uptake or engraftment. Engraftment is measured by assessinggraft versus tumor effect. Other outcomes that are measured includeneutropenic recovery which is assessed by measurement total bloodcounts.

Example 57. Prevention Solid Organ Transplant Rejection in a Rat Model

Recipient LBN rats ranged in weight from 325 to 350 grams. ACI donorweights ranged from 200-250 grams. Test article is administered to ratsusing oral gavage. The treatment started the day before transplant andthe entire treatment period ranged from 12 to 27 days. Control ratsreceived saline. Twenty four hours after test article administration,the heterotopic heart transplant is performed. The hearts aretransplanted using a modification of the technique of Ono and Lindsey(J. of Thoracic and Cardiovascular Surgery, 57, 225-229 (1969). The ratsare palpated daily and asystole defined the day of rejection.

Example 58. Immunomodulation of Solid Organ Transplant Recipients

Here is a method of preventing graft rejection in a recipient of atransplanted solid organ, by administering to said mammalian recipientan effective graft rejection preventative amount of test article.Transplanted solid organ may include a kidney, heart, skin, a lung, aliver, a pancreas, an intestine, an endocrine gland, a bladder, or askeletal muscle. Test article is administered pre-transplant at least 24hours before transplant and after transplant beginning 24 hours aftertransplant and then on day 3, day, 5 and day 15. Patients are closelymonitored for clinical signs and symptoms of transplant rejection aswell as complications such as infections. Stool and blood samples arecollected before transplantation, day 3, day 5, day 15 and day 21 andsubsequently subjected to microbiome analysis. Immune response ismonitored by clinical symptoms as well as biochemical analysis of theserum such as measurement of cytokine levels.

Example 59. Inhibition of Antigen Presenting Cells

Peripheral blood mononuclear cells (PBMC) are prepared by densitygradient centrifugation on Ficoll-Paque (Pharmacia). Aliquots of cellsare frozen in 90% FCS with 10% DMSO and stored in liquid nitrogen. Afterthawing, the cells are ished twice with MSC medium (DMEM with lowglucose and 10% FCS) and re-suspended in assay medium (ISCOVE'S with 25mM Hepes, 1 mM sodium pyruvate, 100 μM non-essential amino acids, 100U/ml penicillin, 100 rag/ml streptomycin, 0.25 rag/ml amphotericin B,5.5×10⁻⁵M 2-mercaptoethanol (all reagents from GibcoBLR) and 5% human ABserum (Sigma, MLR tested)). To prepare monocyte derived dendritic cells(moDCs), PBMCs are plated and adherent fraction is enriched for a.CD11c+ DCs by CD11c MACS sorting. Microbes or microbes preincubated withprebiotics or microbial metabolites to be tested are incubated withCD11c+ moDCs for 4-10 h. Following incubation period, effect of DCmaturation is measured by staining and FACS analysis by looking atmarkers such as CD40, CD80, CD86 PD-L1 and PD-L2. Endocytic capacity ismeasured by FITC-dextran incubation followed by FACS analysis). Cytokineproduction e.g. IL-10, IL-4, IL-12 is analyzed by ELISA or intracellularstaining. Effect on naíve T cell stimulation is analyzed by co-culturedby the moDCs preincubated with test article with naïve T cells isolatedfrom PBMCs as described. T cell activation status is analyzed by surfacestaining followed by FACS analysis for CD3, CD4, CD25.

Example 60. Inhibition of Alloreactivation by a Microbial CompositionUsing Mixed Lymphocyte Reaction (MLR) Assays In Vitro

Peripheral blood mononuclear cells (PBMC) are prepared by densitygradient centrifugation on Ficoll-Paque (Pharmacia). Aliquots of cellsare frozen in 90% FCS with 10% DMSO and stored in liquid nitrogen. Afterthawing, the cells are ished twice with MSC medium (DMEM with lowglucose and 10% FCS) and re-suspended in assay medium (ISCOVE'S with 25mM Hepes, 1 mM sodium pyruvate, 100 μM non-essential amino acids, 100U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B,5.5×10⁻⁵M 2-mercaptoethanol (all reagents from GibcoBLR) and 5% human ABserum (Sigma, MLR tested)). To prepare the T cell-enriched fraction,PBMCs from donor X are depleted of monocytes and B cells byimmunomagnetic negative selection. PBMCs are incubated with mouseanti-human CD19 and CD14 mAbs (no azide/low endotoxin (NA/LE) format)followed by biotin-conjugated goat anti-mouse IgG (multiple adsorption)Ab (all reagents from Pharmingen) and streptavidin microbeads (MiltenyiBiotec). Cells are then separated using a magnetic cell sorter (MACS,Miltenyi Biotec). PBMC from donor Y are X-ray irradiated with 3600 rad(12 min at 70 kV) using Cabinet X ray system (Faxitron X ray, BuffaloGrove, Ill.). To prepare monocyte derived dendritic cells (moDCs), PBMCsare plated and adherent fraction is enriched for a. CD11c+ DCs by CD11cMACS sorting. T cells (15×10⁶/dish) from donor X are cultured in 10 cmtissue culture dishes with PBMC/moDCs (15×106 cells/dish) from donor Yfor 7 days. The cells are incubated at 37° C. in 5% CO₂ atmosphere for 7days. Various concentrations of microbial composition or microbespre-incubated with sugars are added to T cells activated in the MLR for3 days at 37° C. in 5% CO₂ atmosphere. In control cultures activated Tcells are cultured without any test agent. At the end of co-cultureperiod, T cells are recovered. CD8 cells are depleted by negativeimmunomagnetic selection with anti-CD8 MicroBeads (Miltenyi Biotec).Aliquots of cells collected before and after depletion are stained withanti-CD4-PE and anti-CD8-APC antibodies (Caltag) and analyzed by FACS. Tcell activation status is analyzed by surface staining followed by FACSanalysis for CD3, CD4, CD25. Phenotypic analysis for regulatory T celldifferentiation is done by surface staining for CD3, CD4, CD25, CD127and Foxp3 intracellular staining followed by FACS analysis. CytokineAnalysis for various cytokines including IL-6, TNF-alpha is done byELISA or BD™ Cytometric Bead Array. To assess T cell proliferationstatus cultures are pulsed with [H³]TdR (Amersham) (5 Ci/mmol, 1μCi/well) for 18 hours immediately after plating, or incubated for 1, 2,3 or 4 days and then pulsed with [H³]TdR) for an additional 18 hours.Cultures are collected using Harvester 96 (Tomtec), filters are analyzedusing Microbeta Trilux liquid scintillation and luminescence counter (E.G. & G Wallac). To assess T cell proliferation status cultures arepulsed with [H³]TdR (Amersham) (5 Ci/mmol, 1 Ci/well) for 18 hoursimmediately after plating, or incubated for 1, 2, 3 or 4 days and thenpulsed with [H³]TdR) for an additional 18 hours. Cultures are collectedusing Harvester 96 (Tomtec), filters are analyzed using Microbeta Triluxliquid scintillation and luminescence counter (E. G. & G Wallac). Toassess effect on cytotoxic capacity of CD8+ cells, at the end ofco-culture period, CD8+ cells are sorted by MACS. CD8+ T cells are thenco-incubated with target cells such as hepatocytes and ⁵¹Cromium for4-16 h. After incubation period, level of ⁵¹Cromium in supernatant ismeasured to gauge cytotoxic activity.

Example 61. Inhibition of PHA Induced T Cell Proliferation and T CellActivation by a Microbial Composition Using Mixed Lymphocyte Reaction(MLR) Assays In Vitro

5×10⁴ T cells are stimulated with PHA (5 μg/ml) and then test articleadded at various concentrations. Alternatively, T cells are firstincubated with test article and then activated with PHA.T cellactivation status is analyzed by surface staining followed by FACSanalysis for CD3, CD4, CD25. To assess T cell proliferation statuscultures are pulsed with [H³]TdR (Amersham) (5 Ci/mmol, 1 μCi/well) for18 hours immediately after plating, or incubated for 1, 2, 3 or 4 daysand then pulsed with [H³]TdR) for an additional 18 hours. Cultures arecollected using Harvester 96 (Tomtec), filters are analyzed usingMicrobeta Trilux liquid scintillation and luminescence counter (E. G. &G Wallac).

Example 62. Culturing and Banking Bacterial Isolates from Mouse or HumanFeces

Entire stool specimens are collected and homogenized in 1-3 volumes of0.05% peptone using a sterile stainless steel blender with 1-3 volumesof peptone. Approximately 1 gram of the specimen is serially diluted(10-fold) in pre-reduced, anaerobically sterilized (PRAS) dilutionblanks (Anaerobe Systems). A separate ˜1 gram aliquot is weight, driedin a vacuum over, and re-weighed in order to calculate counts on adry-weight basis. To select for Clostridiales bacteria, includingBlautia species, 100 μL of the homogenized stool sample dilution seriesis plated on Brain-Heart Infusion blood agar (SBA, Becton Dickinson)supplemented with 4 μg/mL trimethoprim (Sigma Chemical) and 1 μg/mLsulfamethoxazole (Sigma), Brucella Blood Agar (BAP, Anaeobe Systems),CDC ANA blood agar, (BBL Microbiology Systems), and egg yolk agar (EYA,Anaerobe Systems) (Finegold S M, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe E M, Collins M D,Lawson P A, Summanen P, Baysallar M, Tomzynski T J, Read E, Johnson E,Rolfe R, Nasir P, Shah H, Haake D A, Manning P, Kaul A, 2002.Gastrointestinal microflora studies in late-onset autism. Clin InfectDis 1:35). To select for spore-formers, the dilutions may be heated at70-80° C. for 10-20 minutes and plated in the same manner as thenon-heated homogenized stool samples. After 5 days of growth at 37° C.in an anaerobic chamber, single colonies are selected. The colonypurification process is repeated by restreaking select single colonies,growing as described above, and selecting again for single colonies.Single colonies are frozen in 15%-25% glycerol in 1 mL cryotubes andstored at −80° C.

Example 63. Sampling of Human Vaginal Microflora

The vaginal microflora were collected in duplicate from the left andright sides of the vaginal sidewall using FLOQSwabs® (Copan Diagnostics,USA) (Jacobson J., et al. 2014. Vaginal microbiome changes withlevonorgestrel intrauterine system placement. Contraception. 90(2):130-135). To control for variables that can alter the vaginalmicrobiome, samples were collected at the same time of a woman'smenstrual cycle (i.e., one week into the menstrual cycle) and patientswere tested for pregnancy and for recent sexual activity (using aprostate-specific antigen membrane test).

Example 64. Sampling of Human Lung Microflora

Bronchoscopy was performed using endrotracheal tube (Combicath™, KOLBio-Medical Instruments, USA). Using a syringe, Normal Saline at 1 mg/kgwas lavaged into the right middle lobe of the lung. For adult patients,2-5 mL of bronchoalveolar lavage fluid (BALF) was collected into asterile sputum trap. For children ages three and older, 2 mL. wascollected; 1 mL was collected from children 1-3 years old; 0.5 mL ofBALF was collected from children under the age of one. Within tenminutes of sample collection the sample was transferred from the sterilesputum trap to a sterile container and frozen and stored at −80 C.

Example 65. Preparation of Bacterial Suspension

A human microbiome sample from stool, saliva, or tissue is obtained froma healthy, normal subjects or subjects suffering from a particularcondition which enriches their microbiome for unique and desirablespecies. The sample is diluted to produce a 10-50% slurry insaline+glycerol solution (0.9% (w/v) NaCl, 10% (w/w) glycerol) andplaced in a filter membrane-containing stomaching bag. The material isthen homogenized and removed from the filtered side of the bag producingthe bacterial suspension. Alternatively, a blender is used and filteringis performed after the blending. A low speed centrifugation step is usedas an alternative to filtering to remove the large, non-bacterialcomponents of the suspension. The bacterial suspension is tittered byproducing serial dilutions differing by a log and plating on BBA agarand growing at 37 C in anaerobic conditions. Colonies are consideredcountable at between 10-400 colonies per plate and triplicates areplated for each dilution. The bacterial suspension is flash frozen andstored at −80 C for future use.

Isolation of Spore Formers

To isolate the subpopulation of spore formers, the bacterial slurry istreated with 100% ethanol to generate a 50% ethanol slurry for 1 hr.Alternatively a heat treatment of 50C for 30 minutes is added toinactivate the bacteria that are not capable of forming spores. The 50%ethanol suspension is then pelleted by centrifugation (13,000 rpm for 5min) and the pellet is washed with equal 10× volume of saline and 10%glycerol 3 times to remove the excess ethanol. The final spore fractionis snap frozen in liquid nitrogen in a solution of injection gradesaline and 10% glycerol for subsequent use and stored at −80 C.

Alternatively, a 10% w/v suspension of human fecal material in PBS isincubated in a water bath at 80 degrees Celsius for 30 minutes. Glycerolis added to a final concentration of 15% and then the enriched sporecontaining material is stored at −80 degrees Celsius.

Alternatively, a 10% w/v suspension of human feces in PBS is prepared tocontain a final concentration of 0.5 to 2% Triton X-100. After shakingincubation for 30 minutes at 25 to 37 degrees Celsius, the sample iscentrifuged at 1000 g for 5-10 minutes to pellet particulate matter andlarge cells. The bacterial entities are recovered in the supernatantfraction, where the purified spore population is optionally furthertreated, such as by thermal treatment and/or ethanol treatment asdescribed above.

Example 66. Determining Titer of Bacteria

Counts of viable bacteria are determined by performing 10-fold serialdilutions in PBS and plating to Brucella Blood Agar Petri plates orother applicable solid media known to one skilled in the art (see. e.g.The Manual of Clinical Microbiology ASM Press, 10th Edition or Atlas,Handbook of Microbiological Media, 4th ed, ASM Press, 2010). Plates areincubated at 37 degrees Celsius for 2 days. Colonies are counted from adilution plate with 50-400 colonies and used to back-calculate thenumber of viable bacteria in the population. Visual counts aredetermined by phase contrast microscopy for further morphologicalidentification.

Alternatively, optical density measurements of bacteria containing mediais used to determine a the concentration of bacteria by comparing to astandard curve of known concentrations of bacteria that have previouslymeasured optical densities in culture.

Bacteria are also isolated and quantified by cell sorting techniquesknown to one skilled in the art (e.g. see Nebe-von-Caron, G., Stephens,P. J. & Hewitt, C. J. Analysis of bacterial function by multi-colorfluorescence flow cytometry and single cell sorting. Journal ofMicrobiological Methods, 2000). With this technique surface antibodiesare raised to specific markers of a desired bacteria and they areincubated, washed, and imaged via flow cytometry to count bacteria in acomplex mixture of other bacteria or tissue.

Example 67. FFAB Culturing and Banking Fungal Isolates

Microbial samples e.g. fecal sample; skin swab sample, are washed andhomogenized in PBS. Samples are then serially diluted and fivefolddilutions are spread-plated in triplicate on Sobour and dextrose agar,potato dextrose agar, malt agar, and yeast peptone dextrose (YPD) agareach supplemented with chloramphenicol (40 μg/ml) and kanamycin (50ag/ml). Alternatively, cultures can be grown in liquid conditions onSabouraud Dextrose Broth (SDB; EMD chemicals) at 37° C., 30° C. and 20°C. and subsequently plated to enrich for yeast fractions. Plates areincubated both aerobically and anaerobically for 48 hours at 37° C., 30°C., and 20° C. and subsequent colonies are counted. Random colonies areselected with different morphologies and are re-streaked three times toobtain a pure culture. Cultures can then be grown up in liquid cultureas described above, placed in 10% glycerol and stored at −80° C. forbanking purposes. A sample of culture can be submitted for geneticanalysis by extracting DNA and performing 18S and ITS identification asdescribed herein

Example 68. Measurement of Metabolites with Mass Spectrometry

To determine the prebiotics in a complex media enabling specific growth,fractionation and mass spectrometry techniques are used to identifyprebiotics compounds in media responsible for specific growth.Basically, bacteria are grown in media that has been fractionated byHPLC using standard techniques, and fractions are tested for theirability to promote growth. The fractions that promote growth are furtherfractionated or metabolites in the media are identified using HPLC-MStechniques described below. Furthermore, any metabolomics on tissues,fresh or spent media, blood, or mammalian excretions are determinedusing methods described herein. Unbiased methods exist to determine therelative concentration of metabolites in a sample and are known to oneskilled in the art. Gas or liquid chromatography combined with massspectrometry demonstrate the amounts and identities of variousmetabolites in the aforementioned samples and are further validated byobtaining pure metabolites and running on through the same LC-MSsystems.

Gas Chromatography Mass Spectrometry

Polar metabolites and fatty acids are extracted using monophasic orbiphasic systems of organic solvents and an aqueous sample as previouslydescribed (Metallo et al., 2012, Fendt et al., 2013). Derivatization ofboth polar metabolites and fatty acids has been described previously(Metallo et al., 2012). Briefly, polar metabolites are derivatized toform methoxime-tBDMS derivatives by incubation with 2% methoxylaminehydrochloride (MP Biomedicals) in pyridine (or MOX reagent (ThermoScientific) followed by addition ofN-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1%tert-butyldimethylchlorosilane (t-BDMCS) (Regis Technologies). Non-polarfractions, including triacylglycerides and phospholipids are saponifiedto free fatty acids and esterified to form fatty acid methyl esterseither by incubation with 2% H2SO4 in methanol or by using Methyl-8reagent (Thermo Scientific). Derivatized samples are analysed by GC-MSusing a DB-35MS column (30 m×0.25 mm i.d.×0.25 μm, Agilent J&WScientific) installed in an Agilent 7890A gas chromatograph (GC)interfaced with an Agilent 5975C mass spectrometer (MS). Mass isotopomerdistributions are determined by integrating metabolite ion fragments andcorrected for natural abundance using algorithms adapted from Fernandezet al. (Fernandez et al., 1996).

Liquid Chromatography Mass Spectrometry of Polar Metabolites

After extraction, samples are transferred to a polypropylene vial andsamples are analysed using a Q Exactive Benchtop LC-MS/MS (Thermo FisherScientific). Chromatographic separation is achieved by injecting 2 μL ofsample on a SeQuant ZIC-pHILIC Polymeric column (2.1×150 mm 5 uM, EMDMillipore). Flow rate is set to 100 μL/min, column compartment is set to25C, and autosampler sample tray is set to 4° C. Mobile Phase A consistsof 20 mM Ammonium Carbonate, 0.1% Ammonium Hydroxide in 100% Water.Mobile Phase B is 100% Acetonitrile. The mobile phase gradient (% B) isas follows: 0 min 80%, 5 min 80%, 30 min 20%, 31 min 80%, 42 min 80%.All mobile phase is introduced into the Ion Max source equipped with aHESI II probe set with the following parameters: Sheath Gas=40, AuxGas=15, Sweep Gas=1, Spray Voltage=3.1 kV, Capillary Temperature=275 C,S-lens RF level=40, Heater Temp=350 C. Metabolites are monitored innegative or positive mode using full scan or a targeted selected ionmonitoring (tSIM) method. For tSIM methods, raw counts are corrected forquadropole bias by measuring the quadropole bias experimentally in a setof adjacent runs of samples at natural abundance. Quadropole bias ismeasured for all species by monitoring the measured vs. theoreticalm1/m0 ratio at natural abundance of all species with m-1, m0, m1, and m2centred scans. Quadropole bias-corrected counts are additionallycorrected for natural abundance to obtain the final mass isotopomerdistribution for each compound in each sample.

Example 69. Selection of Oligosaccharides to Augment Gut Microbiome orthe Growth of Administered Microbes

The ability of bacterial isolates to grow on a panel of simple andcomplex carbohydrates is evaluated using a phenotypic array whosecomposition has been previously described previously (Martens E C, LoweE C, Chiang H, Pudlo N A, Wu M, et al. 2011. Recognition and degradationof plant cell wall polysaccharides by two human gut symbionts. PLoS Biol9: e1001221). Bacteria isolates are removed from a frozen stock andgrown in synthetic minimal media overnight and washed in PBS twice toensure minimal transfer of residual materials. They are then grown insynthetic minimal media with various prebiotic substrates as specifiedby the manufacturer. Growth measurements [(optical density at 600 nm(OD)₆₀₀)] are collected every 30 min in duplicate over the course of 3days at 37° C. under anaerobic conditions. A total of three independentexperiments are performed for each species tested (n=6 growthprofiles/substrate/species). Total growth (A_(tot)) is calculated fromeach growth curve as the difference between the maximum and minimumoptical densities (OD₆₀₀) observed (i.e., A_(max)−A_(min)). Growth ratesare calculated as total growth divided by time(A_(tot)/(t_(max)−t_(min))), where t_(max) and t_(min) correspond to thetime-points at which Amax and A_(max) and A_(min), respectively, werecollected.

This may be followed by a step to ensure that the selectedoligosaccharide(s) promote the growth of the healthy-state gutmicrobiota and/or the microbe(s) comprising a therapeutic compositionwithout augmenting the growth of microbes associated with an autoimmuneor inflammatory disease state. By testing oligosaccharides against apanel of bacteria (individually or in groups) overrepresented in aselected autoimmune or inflammatory condition, a prebiotic thatselectively allows enhanced growth of healthy-state bacteria overdisease-state bacteria is selected.

Example 70. Validating Selective Prebiotics Enhance the Growth ofBacteria in the Blood of Mammalian Subjects

Four cohorts of 8, 6-8 week old Balb/c wildtype male mice acquired andfed a normal diet. One cohort is injected with 100 ul of 1E4 CFU/ml ofthe bacterial composition containing 100 ul 0.1 mg/ml prebiotic mixture,the second cohort is injected with 100 ul of 1E4 CFU/ml of the bacterialcomposition alone, the third is injected with 100 ul 0.1 mg/ml of aprebiotic mixture and the final cohort serves as a vehicle controlcohort injected with vehicle via tail vein. The mice cohorts are thenreadily bleed at 1 hr, 2 hrs 4 hrs 6 hrs 12 hrs and 24 hrs after theinitial administration. At 24 hours gross necropsy is performed and theorgans including the lymph nodes, lungs, liver, pancreas, colon,kidneys, esophagus, mammary glands, prostate, bladder, and blood samplesare assessed for the amount of the administered bacteria by qPCR primersdesigned for the bacteria injected. The samples are normalized to thevehicle control and the biodistribution of the bacteria is assesseddemonstrating the ability of the prebiotic to alter the distribution ofthe administered bacteria when compared with the bacteria administeredalone. Additional the experiment is repeated with oral administration ofthe bacteria at 1E10 CFU/ml and prebiotic mixture administered at 10mg/ml via gavage. The prebiotic mixture demonstrates the ability to bothenhance the level of the bacteria observed in blood and other organsincluding the lungs, kidneys, liver, colon, pancreas,

16s analysis is further performed to assess the effects on cohorts ofbacteria not present in bacterial composition administered. Comparingthe combined bacteria and prebiotic composition to the other cohortsshows the enhanced growth observed in a mammalian subject. The blood isalso submitted for metabolomics with the pure prebiotic compositionadministered as a control to demonstrate appropriate utilization andproduction of specific bacterial metabolites not present in compositionscontaining the combination of bacteria and prebiotic.

Example 71. Selection of Immunomodulatory Carbohydrates or FungalSpecies

A carbohydrate library is selected based on production by bacteriaassociated with a healthy microbiome or bacteria associated with adisease state including but not limited to Type 1 Diabetes,Graft-Versus-Host Disease, Crohn's Disease, Celiac Disease, andIrritable Bowel Syndrome. In some embodiments, the carbohydrates arefunctionalized with an amine linker at the reducing end of the sugar anddissolved in phosphate buffer (50 mM NaH₂PO₄, pH 8.5) at a concentrationof 1 mM. In other embodiments. the carbohydrates are functionalized witha thiol linker at the reducing end of the sugar and dissolved in PBS (pH7.4; including an equimolar amount of tris(2-carboxyethyl)phosphinehydrochloride (Thermo Scientific) at a concentration of 1 mM. Thecompounds are robotically printed in triplicates using a piezoelectricspotting device (S3, Scienion) onto epoxy functionalized microarrayslides (sciChip Epoxy, Scienion) in 60% relative humidity, at 23° C. Theslides are placed in a humidified chamber for 18 hours and then storedin an anhydrous environment.

Prior to using the microarray, the slides are washed three times withwater, incubated with 100 mM ethanolamine in 50 mM NaH₂PO₄ buffer (pH 9)at 50° C. for 1 hour, rinsed again three times with water, and finallydried by centrifugation. The microarray slides are blocked with blockingbuffer (10 mM HEPES, 1 mM CaCl₂, 1 mM MgCl₂, 2% BSA) at room temperaturefor 1 hour and washed three times with lectin buffer (10 mM HEPES, 1 mMCaCl₂, 1 mM MgCl₂) for 5 min. 1 μg of C-type lectin receptor-bindingprotein sample, diluted in lectin buffer supplemented with 0.01% Tween20, is applied and the slides are incubated for 1 hour at roomtemperature. The arrays are washed three times with lectin buffer for 5min and monoclonal mouse anti-human IgG1-AlexaFluor 488 (Invitrogen,Carlsbad, Calif.) is applied at a 1:100 dilution in lectin buffer with0.5% BSA at room temperature for 1 hour. The slides are then washedtwice with lectin buffer and once with distilled water, spun at 1000 rpmfor 5 min, and scanned with a Genepix scanner 7 (Molecular Devices,Sunnyvale, Calif., USA). Binding affinities are determined by measuringthe mean fluorescent intensities (MFI) using Genepix Pro 7 (MolecularDevices, Sunnyvale, Calif., USA) (Maglinao M, Eriksson M, Schlegel M K,Zimmermann S, Johannssen T, Gitze S, Seeberger P H, Lepenies B, 2014. Aplatform to screen for C-type lectin receptor-binding carbohydrates andtheir potential for cell-specific targeting and immune modulation,Journal of Controlled Release, 175:36-42). Ligands for C-type lectinreceptors are often fungal in origin, and thus in addition to being aselection mechanism for carbohydrates, this method also serves as aselection mechanism for immunomodulatory fungal species.

Example 72. Co-Culture of Bacteria Plus Prebiotic and Host-Cells andAnalysis of Host Cell Cytokine Response

The following work is done in the presence and absence (as a control) ofone or more selected prebiotic carbohydrates. This assay may be used totest or confirm the ability of a prebiotic-bacterium pair to elicit animmunomodulatory response such that the production or release ofproinflammatory cytokines decreases and/or the production or release ofanti-inflammatory cytokines increases, may be used to evaluate thedifference in cytokine response in the presence or absence of aprebiotic mixture, and/or may be used to evaluate an array of prebioticcandidates. Clostridiales bacteria are obtained from the ATCC orpurified from a human donor and cultured in brain-heart infusion brothat 37° C. The bacteria are harvested by centrifugation (3000 g, 15minutes) after 24 hours of stationary growth. To test the effects ofspores on human intestinal cells and/or human peripheral bloodmononuclear cells (huPBMC), bacteria are first heat killed (95° C., 30minutes) before the centrifugation step. Bacteria (or spores) are washedthree times with 1×PBS (pH 7.2, Gibco BRL) and subsequently diluted toobtain final cell densities of 10⁶ and 10⁷ colony forming units (cfu)/mlin RPMI 1640 medium (Gibco BRL).

Human enterocyte-like CaCO-2 cells (passage 60-65) are seeded at adensity of 2.5×10⁵ cells/ml on 25 mm cell culture inserts (0.4 mnucleopore size; Becton Dickinson). The inserts are placed into six welltissue culture plates (Nunc) and cultured 18-22 days at 37° C./10% CO₂in DMEM (glutamine, high glucose; Amimed) supplemented with 20%decomplemented fetal calf serum (56° C., 30 minutes; Amimed), 1% MEMnon-essential amino acids (Gibco BRL), 10 μg/ml gentamycin (Gibco BRL),and 0.1% penicillin/streptomycin (10 000 IU/ml/10 000 UG/ml; Gibco BRL).The cell culture medium is changed every second day until the cells arefully differentiated. Transepithelial electrical resistance (TEER) isdetermined continuously in confluent CaCO-2 monolayers using aMultiCell-ERS voltmeter/ohmmeter or as described in Example 44.

Tissue culture inserts covered with CaCO-2 cell monolayers are washedtwice with prewarmed RPMI 1640 medium and transferred to six well tissueculture plates. 2 mL culture medium is added to the apical andbasolateral compartments of the transwell cell culture system.

Next, the apical surface of CaCO-2 monolayers is challenged by additionof 10⁶ or 10⁷ cfu/ml of Clostridiales bacteria or spores, in the absenceof gentamicin. After four hours, gentamicin is added (at 150 μg/mL) tostop bacterial growth and metabolite secretion. CaCO-2 cells arestimulated with the bacteria or spores for 6-36 hours in a 37° C., 10%CO₂ incubator. Then the CaCO-2 cells are collected, washed once withcold 1×PBS (pH 7.2), and lysed in denaturation solution for RNAextraction (Micro RNA Isolation Kit, Stratagene). Cellular lysates arestored at −20° C. and cell culture supernatants are collected from theapical compartment and frozen at −20° C. The immune response of CaCO-2cells is monitored by analysis of cytokine gene transcription (TNF-α,IL-8, monocyte chemoattracting protein 1 (MCP-1), TGF-β, IL-12, IFN-γ,IL-4, IL-10) using a reverse transcription-polymerase chain reaction(RT-PCR) technique and determination of cytokine secretion in cellculture supernatants using an ELISA (Haller D, Bode C, Hammes W P,Pfeifer A M A, Schiffrin E J, Blum S, 2000. Non-pathogenic bacteriaelicit a differential cytokine response by intestinal epithelialcell/leucocyte co-cultures. Gut. 47:79-97).

Example 73. Analysis of Microbially-Produced Short Chain Fatty Acids andLactic Acid

Microbes may be selected for administration to a patient based on itsfermentation products. Microbes may be selected for their ability toproduce immunosuppressive short chain fatty acids such as propionate(priopionic acid) and/or butyrate (butyric acid). Such analysis is alsoused to pair microbes with a prebiotic carbohydrate such that theprebiotic carbohydrate is a substrate for the production of the desiredimmunosuppressive fermentation products.

5 M stock solutions of standards [formic acid (FA), acetic acid (AA),propionic acid (PA), butyric acid (BA), valeric acid, iso-caproic acid,D/L-lactic acid (D/L-LA), 2-ethyl-butyric acid and pimelic acid (SigmaAldrich)] are made up in HPLC-grade water (VWR). A 0.2M succinic acid(SA) internal standard is prepared in HPLC-grade water with NaOH (SigmaAldrich) to promote dissolution. Combined working solutions (WS,containing FA, AA, PA, PA, BA and LA) of 0.5 M and 0.05 M are preparedby diluting the stock solution appropriately with HPLC-grade water.Standard solutions of 0.1 M in water/methanol (50/50, v/v) are preparedfor valeric acid, iso-caproic acid, 2-ethyl-butyric acid and pimelicacid.

Microbes are purchased or purified from human donors or patients asdescribed in Examples 18 and 36 and are grown in M2GSC medium. The M2GSCmedium is at pH 6 and contains, per 100 mL:30 mL of rumen fluid, 1 g ofcasitone, 0.25 of yeast extract, 0.2 g of glucose, 0.2 g of cellobiose,0.2 g of soluble starch, 0.045 g of K₂HPO₄, 0.045 g of KH₂PO₄, 0.09 g of(NH₄)₂SO₄, 0.09 g of NaCl, 0.009 g of MgSO₄.7H₂O, 0.009 g of CaCl₂, 0.1mg of resazurin, 0.4 g of NaHCO₃ and 0.1 g of cystein hydrochloride.

For analysis of microbially-produced short chain fatty acids, 1 mLsupernatant from the microbial cultures is placed in a pyrex extractiontube. 50 μL of the SA stock solution is added as an internal standard toeach standard sample or experimental sample. The samples are vortexedand equilibrated at room temperature for 5 minutes. Then, 100 μL ofconcentrated HCl (VWR) is added, followed by vortexing for 15 seconds.The samples are extracted for 20 min by gently rolling using 5 mL ofdiethylether (VWR). After centrifugation (5 min, 3500 rpm). thesupernatant is transferred to another pyrex extraction tube and 500 μLof a 1 M solution of NaOH is added. The samples are extracted again for20 min, followed by a centrifugation step. The aqueous phase istransferred to an autosampler vial and 100 μL of concentrated HCl isadded. After vortexing, a 10 μL aliquot is injected onto the HPLC-UVapparatus, which comprises a P4000 gradient pump with vacuum degassing,an AS3000 autosampler (10° C.). an UV6000 detector, and a SN4000 module(Thermo Separations Products, Thermo Scientific).

Chromatographic separation is performed as described in De Baere S.,Eeckhaut V., Steppe M., De Maesschalck C., De Backer P., Van ImmerseelF., Croubels S., 2013. Development of a HPLC-UV method for thequantitative determination of four short-chain fatty acids and lacticacid produced by intestinal bacteria during in vitro fermentation.Journal of Pharmaceutical and Biomedical Analysis. 80:107-115.

Example 74. Method of Preparing the Microbial and Prebiotic Compositionfor Administration to a Patient

One strain of bacteria or fungi is independently cultured and mixedtogether with a selected prebiotic carbohydrate before administration.The strain is grown at 37° C., pH 7, in a GMM or otheranimal-products-free medium, pre-reduced with 3 g/L cysteineYHCl. Aftereach strain reaches a sufficient biomass, it is preserved for banking byadding 15% glycerol and then frozen at −80° C. in 1 mL cryotubes. Thestrain is then cultivated to a concentration of 10̂10 CFU/mL, thenconcentrated 20-fold by tangential flow microfiltration. The spentmedium is exchanged by diafiltering with a preservative mediumconsisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphatebuffer, or other suitable preservative medium. The suspension isfreeze-dried to a powder and titrated.

After drying, the powder is blended with microcrystalline cellulose andmagnesium stearate and formulated into a 250 mg gelatin capsulecontaining 10 mg of lyophilized powder (10⁸ to 10¹¹ bacteria), 160 mgmicrocrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesiumstearate, and the prebiotic mixture. The prebiotic mixture is in powderform and is mixed with the microbial composition in a ratio ofprebiotic:microbe of about 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10, 1:15,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:70, 1:80, 1:90,1:100, or 1:500.

Example 75. A Rat Model for Radiation- and Chemotherapy-InducedMucositis

Eighteen female Wistar rats of 150-200 grams, aged 14-16 weeks, may beobtained from the Central Animal Research Facility, Manipal University,Manipal (License No. 94/1999 CPCSEA). The rats are housed inpolycarbonate cages and were provided free access to standard rat foodand filtered water. After one week adaptation to the environment, ratsare subjected to chemotherapy by orally administering busulfan(Sigma-Aldrich Co. LLC, St. Louis, Mo., USA) at 6 mg/kg for four days.To administer infrared radiation to the rats, the tail flick apparatus(model 37360, Ugo Basile Srl, Comerio, VA, Italy) is used. The rats areanesthetized with light ether and the dorsal surface of the tongue isexposed to IR radiation of intensity 40 mV/cm² for 5 second on the firstand fourth days of treatment with busulfan (Patel A, Rajesh S.Chandrashekar V M, Rathnam S, Shah K, Rao C M, Nandaku mar K, 2013. Arat model against chemotherapy plus radiation-induced mucositis. SaudiPharmaceutical Journal. 21:399-403).

Example 76. A Mouse Model for Bone Marrow Transplantation

Female C57BL/6 and B6D2F1 mice are purchased from the Jackson Laboratory(Bar Harbor, Me., USA). Bone marrow is harvested from the femurs andtibias of 12-20 week old mice. Before receiving transplantation, B6D2F1mice are given 14 Gy total body irradiation (¹³⁷Cs source). Irradiationis done twice, three hours apart. 5×10̂6 bone marrow cells aresupplemented with 2×10̂6 nylon-wool nonadherent splenetic T cells fromC57BL/6 mice and resuspended in Leibovitz's L15 medium (LifeTechnologies Inc., New York USA) and transplanted by tail vein infusion(0.25 mL) into B6D2F1 mice (Cooke K R, Gerbitz A, Crawford J M, TeshmiaT, Hill G R, Tesolin A. Rossignol D P, Ferrara J L M, 2001. LPSantagonism reduces graft-versus-host disease and preservesgraft-versus-host leukemia after experimental bone marrowtransplantation. The Journal of Clinical Investigation. 107:1581-1589).

Example 77. A Mouse Model for Studying Gut Microbiome in Graft VersusHost Disease

Blood from healthy human donors is collected in a tube containing sodiumheparin. The blood is diluted in an equal volume of Ca²⁺- and Mg²⁺-freephosphate buffered saline with 2% v/v fetal bovine serum and centrifugedat room temperate at 200×g for 10 minutes. The white “buffy coat” layeris removed to yield human peripheral mononuclear cells (huPBMCs), washedfive times in RPMI 1640, and diluted (2×10⁶ cells/mL) in RPMI 1640 withdecomplemented 20% human AB serum (56° C., 30 minutes, Sigma) and 150μg/mL gentamicin.

At 4- to 5 weeks old, the Rag2^(−/−)γc^(−/−) mice (purchased fromTaconic) are pretreated with liposome-clodronate (VU Medisch Centrum)and sublethally irradiated (1 Gy/6 g), then transplantedintraperitoneally with 3.0×10⁷ huPBMCs. After 4 weeks. these humanizedmice sublethally irradiated and, one day later, are injectedintravenously with 1.0×10⁷ allogeneic huPBMCs (1 Gy/6 g). Thetransplanted mice are monitored daily for GVHD symptoms including weightloss, temperature changes, and diarrhea. (Zheng J, Liu Y, Liu U, Liu M,Xiang Z, Lam K-T, Lewis D B, Lau Y-L, Tu W, 2013. Human CD8⁺ RegulatoryT Cells Inhibit GVHD and Preserve General Immunity in Humanized Mice SciTransl Med 5:168ra9.)

Example 78. Detection of Bacteria in Antigen-Presenting Cells

The following methods may be applied to assess the persistence ofdysbiotic or disease-associated bacteria. Optionally, these methods maybe applied to assess immunomodulatory behavior of bacteria administeredto a patient or of bacteria associated with the patient's natural“healthy” microbiome.

Dendritic cells (DCs) are isolated from bone marrow or blood accordingto standard methods or kit protocols (e.g., Inaba K, Swiggard W J,Steinman R M, Romani N, Schuler G, 2001. Isolation of dendritic cells.Current Protocols in Immunology. Chapter 3:Unit3.7) or are obtained fromthe ATCC.

GFP-expressing Clostridiales bacteria are made using the pGLO™ BacterialTransformation Kit (BioRad, USA) according to the manufacturer'sinstructions.

To evaluate bacterial entrance into and/or presence in DCs, 250,000 DCsare seeded on a round cover slip in complete RPMI-1640 medium and arethen infected with GFP-expressing Clostridiales bacteria. After 1 hourof infection, the cells are washed twice with ice-cold PBS. To killextracellular bacteria, the cells are incubated in complete RPMI-1640medium supplemented with 50 μg/ml gentamicin for 2-3 hours. Cells arefixed and permeabilized for 10 min at −20° C. with 100% methanol andblocked for 1 hour with PBS plus 3% bovine serum albumin. Afterincubation, the cover slips is washed three times, mounted on microscopeslides and analyzed on a confocal microscope (e.g., a fluoview FV100Olympus confocal microscope). The number of cells containingintracellular bacteria are counted and normalized to the total number ofcells in the field. Z-stack analysis (using 0.5 am steps) is used todiscern intracellular bacteria from extracellular bacteria (Bueno S M,Wozniak A, Leiva E D, Riquelme S A, Carrefio L J, Hardt W D, Riedel C A,Kalergis A M, 2010. Salmonella pathogenicity island 1 differentiallymodulates bacterial entry to dendritic and non-phagocytic cells.Immunology. 130:273-87). To detect bacteria outside of dendritic cells,confocal microscopy may be performed in which immunospecific antibioticsto the GFP-expressing bacteria are visualized.

Additionally or alternatively, gentamicin protection assays are used toevaluate bacterial survival inside DCs. Overnight cultures ofClostriales bacteria are subcultured until they reached an OD600 of0.5-0.7 and are then washed and resuspended in ice-cold PBS. The DCs areinfected with bacteria a multiplicity of infection (MOI) equal to 25 for1 hour. The DCs are washed and extracellular bacteria are killed byincubating the DCs for 2 hours with 50 μg/ml gentamicin (Sigma-Aldrich).To recover intracellular bacteria, 10,000 live cells are counted andlysed for 15 min with 0.1% Triton-X-100 in PBS. The lysed cells areseeded on Luria-Bertani agar plates and incubated for 12-16 hours at 37°C. to count intracellular bacteria as colony-forming units (CFUs). Datafrom gentamicin protection assays are normalized as the percentage ofrecovered CFUs relative to the maximum amount obtained in eachexperiment (defined as 100%) (Bueno S M, Wozniak A, Leiva E D, RiquelmeS A, Carrefio L J, Hardt W D, Riedel C A, Kalergis A M, 2010. Salmonellapathogenicity island 1 differentially modulates bacterial entry todendritic and non-phagocytic cells. Immunology. 130:273-87). The methodsdescribed above may also be performed in substantially the same manner,using macrophages (obtained from the ATCC) in place of DCs.

Example 79. Detection of Bacteria in MLN, PLN and Spleen

Radiolabeled bacteria may be detected in organs and serve as anindicator of bacterial translocation in an animal model.

To prepare radiolabeled bacteria, the following steps are repeated threetimes: A bacterial sample (e.g., Escherichia coli ATCC-10536) isobtained from the ATCC and grown overnight in an appropriate medium(e.g., trypticasein agar). The next day, the strain is transferred to atube containing 10 mL of sterile saline solution. The bacterialconcentration is adjusted to 11% of transmittance in a spectrophotometerat 580 nm, corresponding to a CFU/mL of approximately 10⁸. Two mL of thebacterial suspension is incubated in tubes containing 1 mL of stannouschloride solution (580 μM, pH 7.0) at 37° C. for 10 minutes. Afterincubation, 37.0-55.5 MBq of ^(99m)Tc obtained by elution from thesterile ⁹⁹Mo/^(99m)Tc generator (IPEN/Brazil) is added, and the sampleis incubated for 10 minutes at 37° C. The tubes are centrifuged at 3000g for 25 minutes.

Once repeated three times, the radioactivity of the supernatant andprecipitate is measured using a dose calibrator (CRC-25R DoseCalibrator; Capintec, Ramsey, N.J.), and the labeling efficiency iscalculated by dividing the cpm of the precipitate by the total cpm(precipitate plus supernatant) and multiplying by 100%.

Adult Swiss male mice are fed standard chow. If the experiment calls forthe assessment of a treatment (e.g., supplementation with citrulline,microbial composition, and/or an immunomodulatory or prebioticcarbohydrate), the mice fed standard chow plus treatment are comparedwith mice only fed standard chow. The mice are administered 0.1 mL of asuspension containing 1.8 MBq of the ^(99m)Tc-labeled bacteria (10⁷CFU/mL), by gavage. One to two days later, the animals were euthanized,and the mesenteric lymph nodes (MLNs), popliteal lymph notes (PLNs), andspleen are removed, weighed, and placed in tubes. Incorporatedradioactivity is assessed using a counter with an NaI (Tl) crystal(ANSR; Abbott, Chicago, Ill.) and normalized to the organ's weight(Batista M A, Nicoli J R, Martins Fdos S, Machado, J A, Arantes R M,Quinino I E, Correia M I, Cardoso V N, 2012. Pretreatment withcitrulline improves gut barrier after intestinal obstruction in mice.JPEN J Parenter Enteral Nutr. 36:69-76).

Example 80. Measuring Intestinal Integrity by Zonulin ELISA

Age-matched male diabetes-prone and diabetes-resistant rats areanesthetized with ketamine and killed at increasing ages (20, 50, 75,and >100 days) by exsanguination. The rat abdominal wall is opened,small intestinal loops are isolated, and intraluminal lavage isperformed by instillation of 0.5 ml of PBS into the proximal smallintestine followed by aspiration. The aspirate is stored at −80° C.until a zonulin EL, ISA is performed as follows. Plastic microtiterplates (Costar, Cambridge, Mass.) are coated with polyclonal rabbit,zonulin-specific anti-Zot antibodies (dilution 1:100) overnight at 4° C.and are then blocked by incubation with 0.05% PBS-Tween 2) for 15 min atroom temperature. A standard curve is made by serial dilution of zonulin(0.78-50 ng/rd) in 0.05% PBS-Tween 20. Equal amounts of the standardsand experimental samples are aliquotted into the microtiter plate wellsand incubated for 1 hour at room temperature. Unbound zonulin is removedby washing, and the wells are incubated by agitation with biotinylatedanti-Zot antibodies for 1 hour at room temperature. A color reaction isdeveloped by adding 100 μl of Extra-Avidin (Sigma) diluted 1:20,000 in0.1 M Tris.HCl, 1 mM MgCl₂, 1% BSA, pH 7.3, for 15 min, followed byincubation with 100 μl of a 1 mg/ml of p-nitrophenyl-phosphate substrate(Sigma) solution. Absorbance at 405 nm is read after 30 min (Watts T,Berti I, Sapone A, Gerarduzzi T, Not T, Zielke R, and Fasano A, 2005.Role of the intestinal tight junction modular zonulin in thepathogenesis of type I diabetes in BB diabetic-prone rats. PNAS.102:2916-2921).

Example 81. Measuring Intestinal Integrity by Western Blot of TightJunction Proteins

Intestinal integrity may also be evaluated by measuring tight junctionprotein levels by Western blot. In this case, primary antibodies foroccluding and zona occludins-1 (Grand Island, N.Y.), primary antibodiesfor claudin-1 and claudin-2 (Santa Cruz Biotechnology, CA), andsecondary antibodies (fluorescein-conjugated goat anti-mouse and goatanti-rabbit from Santa Cruz Biotechnology, CA) are used. One centimetersections of mid-jujunal intestine are harvested from an appropriateanimal model (e.g., rat or mouse) and immediately homogenized in 1 mLice-cold RIPA-buffer (50 mM TRIS-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5mM EDTA, 1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mMphenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 2 μg/ml leupeptin and2 mM sodium orthovanadate). Tissue lysates are sonicated, incubated onice for 20 min and centrifuged at 4° C., and the resulting supernatantsare collected for immunoprecipitation. The protein concentration of thesupernatants is measured using the techniques known to one skilled inthe art, including but not limited to a Bradford assay. The samples areboiled for 5 min and then 2 μg of protein from each sample is loadedinto the lanes of a 10% acrylamide gel. Following electrophoresis, theproteins are transferred onto nitrocellulose filters, which are thenincubated with primary antibodies directed against occludin,zona-occludin-1, claudin-1, and/or claudin-2 overnight at 4° C. Thefilters are then washed with 1×TBST and incubated with secondaryantibodies conjugated with horseradish peroxidase (HRP) for one hour atroom temperature. Immunocomplexes for each of the tight junctionproteins are then detected by chemiluminescence (Alaish S M, Smith A D,Timmons J, Greenspon J, Eyvazzadeh D, Murphy E, Shea-Donahue T,Cirimotich S, Mongodin E, Zhao A, Fasano A, Nataro J P, Cross A, 2013.Gut microbiota, tight junction protein expression, intestinalresistance, bacterial translocation and mortality following cholestasisdepend on the genetic background of the host. Gut Microbes. 4:292-305).

Example 82. Measurement of Intestinal Permeability in a Mouse Model forAlcoholism

Eight-week-old male C57BL/6N mice are fed a modified Lieber-DeCarliliquid diet containing ethanol (35% of total calories) or containing noethanol for one week and then gradually increasing amounts of ethanol(to a maximum of 35% total calories) over the course of β-4 days. Aftereight weeks of ethanol feeding, the mice are fasted overnight,anesthetized intraperitonially with sodium-pentobarbital (nembutal, 80mg/kg), and and whole intestinal samples are collected and weighed. Thefreshly isolated intestinal segments (duodenum, jejunum, ileum) areplaced in Krebs-Henseleit bicarbonate buffer and then used for ex vivointestinal permeability assay as follows. One end of the gut segment isligated with suture, and 200 μl of fluorescent dextran-FITC (FD-4; M.W.4,000, 40 mg/ml) is injected into the lumen using a gavage needle. Theother end of the gut segment is ligated to form a gut sac. The gut sacis rinse in Krebs-Henseleit bicarbonate buffer and placed in 4 ml offresh buffer, then incubated at 37° C. for 20 minutes. The FD-4 thatpenetrated from the lumen into the buffer is measured with aspectrofluorometer using an excitation wave length of 485 nm and anemission wave length of 530 nm (Kirpich I A, Feng W, Wang Y, Lie Y,Barker D F, Barve S S, McClain C J, 2011. The Type of Dietary FatModulates Intestinal Tight Junction Integrity, Gut Permeability, andHepatic Toll-Like Receptor Expression in a Mouse Model of AlcoholicLiver Disease. Alcoholism: Clinical and Experimental Research.36:835-846).

Example 83. Detection of Bacteria in Distal Organs

A fragment from a liver or spleen biopsy 15 minutes after reperfusion ismoved to a sterile tube containing thioglycolate and the immediatelycultured on a medium including but not limited to blood agar, blood agarsupplemented for anaerobes, chocolate blood agar, MacConkey agar, andSabouraud agar. Bacteria are purified as described (Example 18) or usingother medium as described in Example 36 and 16S rDNA sequencing isperformed as described (Examples 7, 31, 62-65).

Example 84: Distal Effects of Microbiota

It has been It has been determined that the presence of certainprobiotics in the microbiome plays a role in the therapeuticeffectiveness of certain immunomodulatory cancer therapies, includinganti-PD-1 and anti-CTLA-4 antibodies. In particular, it has been foundthat the addition of certain bacteria to the gut of a subject enhancesthe activity of these checkpoint inhibitors, resulting in increasedanti-tumor T cell responses and inhibition of tumor growth (See Vétizou,et al. Science, Nov. 5, 2015, science.aad1329 and Sivan et al. Science,Nov. 5, 2015, science.aac4255).

In particular, it has been shown that oral administration ofBifidobacterium or Bacteroides probiotics, but not Lactobacillus,increases the responsiveness to checkpoint inhibitor therapy in cancer(Sivan et al., Vetizou et al). Anti-PD-1 and anti-CTLA-4 antibodytherapies relieve a block, or checkpoint, that otherwise limitsanti-tumor immunity. Checkpoint inhibition by these antibodies resultsin an increase in anti-tumor T cell immune responses, and more effectivekilling of cancer cells. Tumors have also been shown to be surrounded bymicrobiomes that are different from the microbiome in normal adjacenttissues, as a result of the interaction between the immune system andthe cancer. The observation that addition of certain bacteria to the gutleads to an effect on the immune system and on the growth of tumors at adistal site suggests a concomitant impact on the tumor microbiome atthese distal site(s).

In order for the immune cells induced by the combination of a checkpointinhibitor and certain gut bacteria to have an effect, the cells musttravel to the tumor site, altering both the immune and tumorenvironments, which will alter the tumor microbiome as well. Thus, thepresence of bacteria introduced in the gut leads to an alteration in thethe microbiome of tumors at a distal site. The above studies support thedistal effects of microbials, and the role of certain bacteria in theeffectiveness of certain cancer therapies.

REFERENCES

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TABLE 1 See, e.g., WO2014/121304 SEQ ID Public DB Spore Pathogen OTUNumber Accession Clade Former Status Eubacterium saburreum 858 AB525414clade_178 Y N Eubacterium sp. oral clone IR009 866 AY349376 clade_178 YN Lachnospiraceae bacterium ICM62 1061 HQ616401 clade_178 Y NLachnospiraceae bacterium MSX33 1062 HQ616384 clade_178 Y NLachnospiraceae bacterium oral taxon 107 1063 ADDS01000069 clade_178 Y NAlicyclobacillus acidocaldarius 122 NR_074721 clade_179 Y N Clostridiumbaratii 555 NR_029229 clade_223 Y N Clostridium colicanis 576 FJ957863clade_223 Y N Clostridium paraputrificum 611 AB536771 clade_223 Y NClostridium sardiniense 621 NR_041006 clade_223 Y N Eubacterium budayi837 NR_024682 clade_223 Y N Eubacterium moniliforme 851 HF558373clade_223 Y N Eubacterium multiforme 852 NR_024683 clade_223 Y NEubacterium nitritogenes 853 NR_024684 clade_223 Y N Anoxybacillusflavithermus 173 NR_074667 clade_238 Y N Bacillus aerophilus 196NR_042339 clade_238 Y N Bacillus aestuarii 197 GQ980243 clade_238 Y NBacillus amyloliquefaciens 199 NR_075005 clade_238 Y N Bacillusanthracis 200 AAEN01000020 clade_238 Y Category-A Bacillus atrophaeus201 NR_075016 clade_238 Y OP Bacillus badius 202 NR_036893 clade_238 YOP Bacillus cereus 203 ABDJ01000015 clade_238 Y OP Bacillus circulans204 AB271747 clade_238 Y OP Bacillus firmus 207 NR_025842 clade_238 Y OPBacillus flexus 208 NR_024691 clade_238 Y OP Bacillus fordii 209NR_025786 clade_238 Y OP Bacillus halmapalus 211 NR_026144 clade_238 YOP Bacillus herbersteinensis 213 NR_042286 clade_238 Y OP Bacillusidriensis 215 NR_043268 clade_238 Y OP Bacillus lentus 216 NR_040792clade_238 Y OP Bacillus licheniformis 217 NC_006270 clade_238 Y OPBacillus megaterium 218 GU252124 clade_238 Y OP Bacillus nealsonii 219NR_044546 clade_238 Y OP Bacillus niabensis 220 NR_043334 clade_238 Y OPBacillus niacini 221 NR_024695 clade_238 Y OP Bacillus pocheonensis 222NR_041377 clade_238 Y OP Bacillus pumilus 223 NR_074977 clade_238 Y OPBacillus safensis 224 JQ624766 clade_238 Y OP Bacillus simplex 225NR_042136 clade_238 Y OP Bacillus sonorensis 226 NR_025130 clade_238 YOP Bacillus sp. 10403023 MM10403188 227 CAET01000089 clade_238 Y OPBacillus sp. 2_A_57_CT2 230 ACWD01000095 clade_238 Y OP Bacillus sp.2008724126 228 GU252108 clade_238 Y OP Bacillus sp. 2008724139 229GU252111 clade_238 Y OP Bacillus sp. 7_16AIA 231 FN397518 clade_238 Y OPBacillus sp. AP8 233 JX101689 clade_238 Y OP Bacillus sp. B27(2008) 234EU362173 clade_238 Y OP Bacillus sp. BT1B_CT2 235 ACWC01000034 clade_238Y OP Bacillus sp. GB1.1 236 FJ897765 clade_238 Y OP Bacillus sp. GB9 237FJ897766 clade_238 Y OP Bacillus sp. HU19.1 238 FJ897769 clade_238 Y OPBacillus sp. HU29 239 FJ897771 clade_238 Y OP Bacillus sp. HU33.1 240FJ897772 clade_238 Y OP Bacillus sp. JC6 241 JF824800 clade_238 Y OPBacillus sp. oral taxon F79 248 HM099654 clade_238 Y OP Bacillus sp.SRC_DSF1 243 GU797283 clade_238 Y OP Bacillus sp. SRC_DSF10 242 GU797292clade_238 Y OP Bacillus sp. SRC_DSF2 244 GU797284 clade_238 Y OPBacillus sp. SRC_DSF6 245 GU797288 clade_238 Y OP Bacillus sp. tc09 249HQ844242 clade_238 Y OP Bacillus sp. zh168 250 FJ851424 clade_238 Y OPBacillus sphaericus 251 DQ286318 clade_238 Y OP Bacillussporothermodurans 252 NR_026010 clade_238 Y OP Bacillus subtilis 253EU627588 clade_238 Y OP Bacillus thermoamylovorans 254 NR_029151clade_238 Y OP Bacillus thuringiensis 255 NC_008600 clade_238 Y OPBacillus weihenstephanensis 256 NR_074926 clade_238 Y OP Geobacilluskaustophilus 933 NR_074989 clade_238 Y N Geobacillus stearothermophilus936 NR_040794 clade_238 Y N Geobacillus thermodenitrificans 938NR_074976 clade_238 Y N Geobacillus thermoglucosidasius 939 NR_043022clade_238 Y N Lysinibacillus sphaericus 1193 NR_074883 clade_238 Y NClostridiales sp. SS3_4 543 AY305316 clade_246 Y N Clostridiumbeijerinckii 557 NR_074434 clade_252 Y N Clostridium botulinum 560NC_010723 clade_252 Y Category-A Clostridium butyricum 561 ABDT01000017clade_252 Y N Clostridium chauvoei 568 EU106372 clade_252 Y NClostridium favososporum 582 X76749 clade_252 Y N Clostridiumhistolyticum 592 HF558362 clade_252 Y N Clostridium isatidis 597NR_026347 clade_252 Y N Clostridium limosum 602 FR870444 clade_252 Y NClostridium sartagoforme 622 NR_026490 clade_252 Y N Clostridiumsepticum 624 NR_026020 clade_252 Y N Clostridium sp. 7_2_43FAA 626ACDK01000101 clade_252 Y N Clostridium sporogenes 645 ABKW02000003clade_252 Y N Clostridium tertium 653 Y18174 clade_252 Y N Clostridiumcarnis 564 NR_044716 clade_253 Y N Clostridium celatum 565 X77844clade_253 Y N Clostridium disporicum 579 NR_026491 clade_253 Y NClostridium gasigenes 585 NR_024945 clade_253 Y N Clostridium quinii 616NR_026149 clade_253 Y N Clostridium hylemonae 593 AB023973 clade_260 Y NClostridium scindens 623 AF262238 clade_260 Y N Lachnospiraceaebacterium 5_1_57FAA 1054 ACTR01000020 clade_260 Y N Clostridiumglycyrrhizinilyticum 588 AB233029 clade_262 Y N Clostridium nexile 607X73443 clade_262 Y N Coprococcus comes 674 ABVR01000038 clade_262 Y NLachnospiraceae bacterium 1_1_57FAA 1048 ACTM01000065 clade_262 Y NLachnospiraceae bacterium 1_4_56FAA 1049 ACTN01000028 clade_262 Y NLachnospiraceae bacterium 8_1_57FAA 1057 ACWQ01000079 clade_262 Y NRuminococcus lactaris 1663 ABOU02000049 clade_262 Y N Ruminococcustorques 1670 AAVP02000002 clade_262 Y N Paenibacillus lautus 1397NR_040882 clade_270 Y N Paenibacillus polymyxa 1399 NR_037006 clade_270Y N Paenibacillus sp. HGF5 1402 AEXS01000095 clade_270 Y N Paenibacillussp. HGF7 1403 AFDH01000147 clade_270 Y N Eubacterium sp. oral cloneJI012 868 AY349379 clade_298 Y N Alicyclobacillus contaminans 124NR_041475 clade_301 Y N Alicyclobacillus herbarius 126 NR_024753clade_301 Y N Alicyclobacillus pomorum 127 NR_024801 clade_301 Y NBlautia coccoides 373 AB571656 clade_309 Y N Blautia glucerasea 374AB588023 clade_309 Y N Blautia glucerasei 375 AB439724 clade_309 Y NBlautia hansenii 376 ABYU02000037 clade_309 Y N Blautia luti 378AB691576 clade_309 Y N Blautia producta 379 AB600998 clade_309 Y NBlautia schinkii 380 NR_026312 clade_309 Y N Blautia sp. M25 381HM626178 clade_309 Y N Blautia stercoris 382 HM626177 clade_309 Y NBlautia wexlerae 383 EF036467 clade_309 Y N Bryantella formatexigens 439ACCL02000018 clade_309 Y N Clostridium coccoides 573 EF025906 clade_309Y N Eubacterium cellulosolvens 839 AY178842 clade_309 Y NLachnospiraceae bacterium 6_1_63FAA 1056 ACTV01000014 clade_309 Y NRuminococcus hansenii 1662 M59114 clade_309 Y N Ruminococcus obeum 1664AY169419 clade_309 Y N Ruminococcus sp. 5_1_39BFAA 1666 ACII01000172clade_309 Y N Ruminococcus sp. K_1 1669 AB222208 clade_309 Y NSyntrophococcus sucromutans 1911 NR_036869 clade_309 Y N Bacillusalcalophilus 198 X76436 clade_327 Y N Bacillus clausii 205 FN397477clade_327 Y OP Bacillus gelatini 210 NR_025595 clade_327 Y OP Bacillushalodurans 212 AY144582 clade_327 Y OP Bacillus sp. oral taxon F26 246HM099642 clade_327 Y OP Clostridium innocuum 595 M23732 clade_351 Y NClostridium sp. HGF2 628 AENW01000022 clade_351 Y N Clostridiumperfringens 612 ABDW01000023 clade_353 Y Category-B Sarcina ventriculi1687 NR_026146 clade_353 Y N Clostridium bartlettii 556 ABEZ02000012clade_354 Y N Clostridium bifermentans 558 X73437 clade_354 Y NClostridium ghonii 586 AB542933 clade_354 Y N Clostridium glycolicum 587FJ384385 clade_354 Y N Clostridium mayombei 605 FR733682 clade_354 Y NClostridium sordellii 625 AB448946 clade_354 Y N Clostridium sp. MT4 E635 FJ159523 clade_354 Y N Eubacterium tenue 872 M59118 clade_354 Y NClostridium argentinense 553 NR_029232 clade_355 Y N Clostridium sp.JC122 630 CAEV01000127 clade_355 Y N Clostridium sp. NMBHI_1 636JN093130 clade_355 Y N Clostridium subterminale 650 NR_041795 clade_355Y N Clostridium sulfidigenes 651 NR_044161 clade_355 Y N Doreaformicigenerans 773 AAXA02000006 clade_360 Y N Dorea longicatena 774AJ132842 clade_360 Y N Lachnospiraceae bacterium 2_1_46FAA 1050ADLB01000035 clade_360 Y N Lachnospiraceae bacterium 2_1_58FAA 1051ACTO01000052 clade_360 Y N Lachnospiraceae bacterium 4_1_37FAA 1053ADCR01000030 clade_360 Y N Lachnospiraceae bacterium 9_1_43BFAA 1058ACTX01000023 clade_360 Y N Ruminococcus gnavus 1661 X94967 clade_360 Y NRuminococcus sp. ID8 1668 AY960564 clade_360 Y N Blautiahydrogenotrophica 377 ACBZ01000217 clade_368 Y N Lactonifactorlongoviformis 1147 DQ100449 clade_368 Y N Robinsoniella peoriensis 1633AF445258 clade_368 Y N Eubacterium infirmum 849 U13039 clade_384 Y NEubacterium sp. WAL 14571 864 FJ687606 clade_384 Y N Erysipelotrichaceaebacterium 5_2_54FAA 823 ACZW01000054 clade_385 Y N Eubacterium biforme835 ABYT01000002 clade_385 Y N Eubacterium cylindroides 842 FP929041clade_385 Y N Eubacterium dolichum 844 L34682 clade_385 Y N Eubacteriumsp. 3_1_31 861 ACTL01000045 clade_385 Y N Eubacterium tortuosum 873NR_044648 clade_385 Y N Bulleidia extructa 441 ADFR01000011 clade_388 YN Solobacterium moorei 1739 AECQ01000039 clade_388 Y N Coprococcus catus673 EU266552 clade_393 Y N Lachnospiraceae bacterium oral taxon F15 1064HM099641 clade_393 Y N Clostridium cochlearium 574 NR_044717 clade_395 YN Clostridium malenominatum 604 FR749893 clade_395 Y N Clostridiumtetani 654 NC_004557 clade_395 Y N Acetivibrio ethanolgignens 6 FR749897clade_396 Y N Anaerosporobacter mobilis 161 NR_042953 clade_396 Y NBacteroides pectinophilus 288 ABVQ01000036 clade_396 Y N Clostridiumaminovalericum 551 NR_029245 clade_396 Y N Clostridium phytofermentans613 NR_074652 clade_396 Y N Eubacterium hallii 848 L34621 clade_396 Y NEubacterium xylanophilum 875 L34628 clade_396 Y N Ruminococcus callidus1658 NR_029160 clade_406 Y N Ruminococcus champanellensis 1659 FP929052clade_406 Y N Ruminococcus sp. 18P13 1665 AJ515913 clade_406 Y NRuminococcus sp. 9SE51 1667 FM954974 clade_406 Y N Anaerostipes caccae162 ABAX03000023 clade_408 Y N Anaerostipes sp. 3_2_56FAA 163ACWB01000002 clade_408 Y N Clostridiales bacterium 1_7_47FAA 541ABQR01000074 clade_408 Y N Clostridiales sp. SM4_1 542 FP929060clade_408 Y N Clostridiales sp. SSC_2 544 FP929061 clade_408 Y NClostridium aerotolerans 546 X76163 clade_408 Y N Clostridium aldenense547 NR_043680 clade_408 Y N Clostridium algidixylanolyticum 550NR_028726 clade_408 Y N Clostridium amygdalinum 552 AY353957 clade_408 YN Clostridium asparagiforme 554 ACCJ01000522 clade_408 Y N Clostridiumbolteae 559 ABCC02000039 clade_408 Y N Clostridium celerecrescens 566JQ246092 clade_408 Y N Clostridium citroniae 569 ADLJ01000059 clade_408Y N Clostridium clostridiiformes 571 M59089 clade_408 Y N Clostridiumclostridioforme 572 NR_044715 clade_408 Y N Clostridium hathewayi 590AY552788 clade_408 Y N Clostridium indolis 594 AF028351 clade_408 Y NClostridium lavalense 600 EF564277 clade_408 Y N Clostridiumsaccharolyticum 620 CP002109 clade_408 Y N Clostridium sp. M62_1 633ACFX02000046 clade_408 Y N Clostridium sp. SS2_1 638 ABGC03000041clade_408 Y N Clostridium sphenoides 643 X73449 clade_408 Y NClostridium symbiosum 652 ADLQ01000114 clade_408 Y N Clostridiumxylanolyticum 658 NR_037068 clade_408 Y N Eubacterium hadrum 847FR749933 clade_408 Y N Lachnospiraceae bacterium 3_1_57FAA_CT1 1052ACTP01000124 clade_408 Y N Lachnospiraceae bacterium 5_1_63FAA 1055ACTS01000081 clade_408 Y N Lachnospiraceae bacterium A4 1059 DQ789118clade_408 Y N Lachnospiraceae bacterium DJF VP30 1060 EU728771 clade_408Y N Lachnospiraceae genomosp. C1 1065 AY278618 clade_408 Y N Clostridiumdifficile 578 NC_013315 clade_409 Y OP Eubacterium sp. AS15b 862HQ616364 clade_428 Y N Eubacterium sp. OBRC9 863 HQ616354 clade_428 Y NEubacterium sp. oral clone OH3A 871 AY947497 clade_428 Y N Eubacteriumyurii 876 AEES01000073 clade_428 Y N Clostridium acetobutylicum 545NR_074511 clade_430 Y N Clostridium algidicarnis 549 NR_041746 clade_430Y N Clostridium cadaveris 562 AB542932 clade_430 Y N Clostridiumcarboxidivorans 563 FR733710 clade_430 Y N Clostridium estertheticum 580NR_042153 clade_430 Y N Clostridium fallax 581 NR_044714 clade_430 Y NClostridium felsineum 583 AF270502 clade_430 Y N Clostridiumfrigidicarnis 584 NR_024919 clade_430 Y N Clostridium kluyveri 598NR_074165 clade_430 Y N Clostridium magnum 603 X77835 clade_430 Y NClostridium putrefaciens 615 NR_024995 clade_430 Y N Clostridium sp.HPB_46 629 AY862516 clade_430 Y N Clostridium tyrobutyricum 656NR_044718 clade_430 Y N Sutterella parvirubra 1899 AB300989 clade_432 YN Acetanaerobacterium elongatum 4 NR_042930 clade_439 Y N Clostridiumcellulosi 567 NR_044624 clade_439 Y N Ethanoligenens harbinense 832AY675965 clade_439 Y N Eubacterium rectale 856 FP929042 clade_444 Y NEubacterium sp. oral clone GI038 865 AY349374 clade_444 Y NLachnobacterium bovis 1045 GU324407 clade_444 Y N Roseburia cecicola1634 GU233441 clade_444 Y N Roseburia faecalis 1635 AY804149 clade_444 YN Roseburia faecis 1636 AY305310 clade_444 Y N Roseburia hominis 1637AJ270482 clade_444 Y N Roseburia intestinalis 1638 FP929050 clade_444 YN Roseburia inulinivorans 1639 AJ270473 clade_444 Y N Brevibacillusbrevis 410 NR_041524 clade_448 Y N Brevibacillus laterosporus 414NR_037005 clade_448 Y N Bacillus coagulans 206 DQ297928 clade_451 Y OPSporolactobacillus inulinus 1752 NR_040962 clade_451 Y N Kocuriapalustris 1041 EU333884 clade_453 Y N Nocardia farcinica 1353 NC_006361clade_455 Y N Bacillus sp. oral taxon F28 247 HM099650 clade_456 Y OPCatenibacterium mitsuokai 495 AB030224 clade_469 Y N Clostridium sp.TM_40 640 AB249652 clade_469 Y N Coprobacillus cateniformis 670 AB030218clade_469 Y N Coprobacillus sp. 29_1 671 ADKX01000057 clade_469 Y NClostridium rectum 618 NR_029271 clade_470 Y N Eubacterium nodatum 854U13041 clade_476 Y N Eubacterium saphenum 859 NR_026031 clade_476 Y NEubacterium sp. oral clone JH012 867 AY349373 clade_476 Y N Eubacteriumsp. oral clone JS001 870 AY349378 clade_476 Y N Faecalibacteriumprausnitzii 880 ACOP02000011 clade_478 Y N Gemmiger formicilis 932GU562446 clade_478 Y N Subdoligranulum variabile 1896 AJ518869 clade_478Y N Clostridiaceae bacterium JC13 532 JF824807 clade_479 Y N Clostridiumsp. MLG055 634 AF304435 clade_479 Y N Erysipelotrichaceae bacterium3_1_53 822 ACTJ01000113 clade_479 Y N Clostridium cocleatum 575NR_026495 clade_481 Y N Clostridium ramosum 617 M23731 clade_481 Y NClostridium saccharogumia 619 DQ100445 clade_481 Y N Clostridiumspiroforme 644 X73441 clade_481 Y N Coprobacillus sp. D7 672ACDT01000199 clade_481 Y N Clostridiales bacterium SY8519 535 AB477431clade_482 Y N Clostridium sp. SY8519 639 AP012212 clade_482 Y NEubacterium ramulus 855 AJ011522 clade_482 Y N Erysipelothrix inopinata819 NR_025594 clade_485 Y N Erysipelothrix rhusiopathiae 820ACLK01000021 clade_485 Y N Erysipelothrix tonsillarum 821 NR_040871clade_485 Y N Holdemania filiformis 1004 Y11466 clade_485 Y N Mollicutesbacterium pACH93 1258 AY297808 clade_485 Y N Coxiella burnetii 736CP000890 clade_486 Y Category-B Clostridium hiranonis 591 AB023970clade_487 Y N Clostridium irregulare 596 NR_029249 clade_487 Y NClostridium orbiscindens 609 Y18187 clade_494 Y N Clostridium sp. NML04A032 637 EU815224 clade_494 Y N Flavonifractor plautii 886 AY724678clade_494 Y N Pseudoflavonifractor capillosus 1591 AY136666 clade_494 YN Ruminococcaceae bacterium D16 1655 ADDX01000083 clade_494 Y NAcetivibrio cellulolyticus 5 NR_025917 clade_495 Y N Clostridiumaldrichii 548 NR_026099 clade_495 Y N Clostridium clariflavum 570NR_041235 clade_495 Y N Clostridium stercorarium 647 NR_025100 clade_495Y N Clostridium straminisolvens 649 NR_024829 clade_495 Y N Clostridiumthermocellum 655 NR_074629 clade_495 Y N Fusobacterium nucleatum 901ADVK01000034 clade_497 Y N Eubacterium barkeri 834 NR_044661 clade_512 YN Eubacterium callanderi 838 NR_026330 clade_512 Y N Eubacterium limosum850 CP002273 clade_512 Y N Anaerotruncus colihominis 164 ABGD02000021clade_516 Y N Clostridium methylpentosum 606 ACEC01000059 clade_516 Y NClostridium sp. YIT 12070 642 AB491208 clade_516 Y NHydrogenoanaerobacterium saccharovorans 1005 NR_044425 clade_516 Y NRuminococcus albus 1656 AY445600 clade_516 Y N Ruminococcus flavefaciens1660 NR_025931 clade_516 Y N Clostridium haemolyticum 589 NR_024749clade_517 Y N Clostridium novyi 608 NR_074343 clade_517 Y N Clostridiumsp. LMG 16094 632 X95274 clade_517 Y N Eubacterium ventriosum 874 L34421clade_519 Y N Bacteroides galacturonicus 280 DQ497994 clade_522 Y NEubacterium eligens 845 CP001104 clade_522 Y N Lachnospira multipara1046 FR733699 clade_522 Y N Lachnospira pectinoschiza 1047 L14675clade_522 Y N Lactobacillus rogosae 1114 GU269544 clade_522 Y N Bacillushorti 214 NR_036860 clade_527 Y OP Bacillus sp. 9_3AIA 232 FN397519clade_527 Y OP Eubacterium brachy 836 U13038 clade_533 Y N Filifactoralocis 881 CP002390 clade_533 Y N Filifactor villosus 882 NR_041928clade_533 Y N Clostridium leptum 601 AJ305238 clade_537 Y N Clostridiumsp. YIT 12069 641 AB491207 clade_537 Y N Clostridium sporosphaeroides646 NR_044835 clade_537 Y N Eubacterium coprostanoligenes 841 HM037995clade_537 Y N Ruminococcus bromii 1657 EU266549 clade_537 Y NEubacterium siraeum 860 ABCA03000054 clade_538 Y N Clostridium viride657 NR_026204 clade_540 Y N Oscillibacter sp. G2 1386 HM626173 clade_540Y N Oscillibacter valericigenes 1387 NR_074793 clade_540 Y NOscillospira guilliermondii 1388 AB040495 clade_540 Y N Butyrivibriocrossotus 455 ABWN01000012 clade_543 Y N Clostridium sp. L2_50 631AAYW02000018 clade_543 Y N Coprococcus eutactus 675 EF031543 clade_543 YN Coprococcus sp. ART55_1 676 AY350746 clade_543 Y N Eubacteriumruminantium 857 NR_024661 clade_543 Y N Collinsella aerofaciens 659AAVN02000007 clade_553 Y N Alkaliphilus metalliredigenes 137 AY137848clade_554 Y N Alkaliphilus oremlandii 138 NR_043674 clade_554 Y NClostridium sticklandii 648 L04167 clade_554 Y N Turicibacter sanguinis1965 AF349724 clade_555 Y N Fulvimonas sp. NML 060897 892 EF589680clade_557 Y N Desulfitobacterium frappieri 753 AJ276701 clade_560 Y NDesulfitobacterium hafniense 754 NR_074996 clade_560 Y NDesulfotomaculum nigrificans 756 NR_044832 clade_560 Y N Lutisporathermophila 1191 NR_041236 clade_564 Y N Brachyspira pilosicoli 405NR_075069 clade_565 Y N Eggerthella lenta 778 AF292375 clade_566 Y NStreptomyces albus 1888 AJ697941 clade_566 Y N Chlamydiales bacteriumNS11 505 JN606074 clade_567 Y N Anaerofustis stercorihominis 159ABIL02000005 clade_570 Y N Butyricicoccus pullicaecorum 453 HH793440clade_572 Y N Eubacterium desmolans 843 NR_044644 clade_572 Y NPapillibacter cinnamivorans 1415 NR_025025 clade_572 Y N Sporobactertermitidis 1751 NR_044972 clade_572 Y N Deferribacteres sp. oral cloneJV006 744 AY349371 clade_575 Y N Clostridium colinum 577 NR_026151clade_576 Y N Clostridium lactatifermentans 599 NR_025651 clade_576 Y NClostridium piliforme 614 D14639 clade_576 Y N Saccharomonospora viridis1671 X54286 clade_579 Y N Thermobifida fusca 1921 NC_007333 clade_579 YN Leptospira licerasiae 1164 EF612284 clade_585 Y OP Moorellathermoacetica 1259 NR_075001 clade_590 Y N Thermoanaerobacterpseudethanolicus 1920 CP000924 clade_590 Y N Flexistipes sinusarabici888 NR_074881 clade_591 Y N Gloeobacter violaceus 942 NR_074282clade_596 Y N Eubacterium sp. oral clone JN088 869 AY349377 clade_90 Y NClostridium oroticum 610 FR749922 clade_96 Y N Clostridium sp. D5 627ADBG01000142 clade_96 Y N Eubacterium contortum 840 FR749946 clade_96 YN Eubacterium fissicatena 846 FR749935 clade_96 Y N Corynebacteriumcoyleae 692 X96497 clade_100 N N Corynebacterium mucifaciens 711NR_026396 clade_100 N N Corynebacterium ureicelerivorans 733 AM397636clade_100 N N Corynebacterium appendicis 684 NR_028951 clade_102 N NCorynebacterium genitalium 698 ACLJ01000031 clade_102 N NCorynebacterium glaucum 699 NR_028971 clade_102 N N Corynebacteriumimitans 703 AF537597 clade_102 N N Corynebacterium riegelii 719 EU848548clade_102 N N Corynebacterium sp. L_2012475 723 HE575405 clade_102 N NCorynebacterium sp. NML 93_0481 724 GU238409 clade_102 N NCorynebacterium sundsvallense 728 Y09655 clade_102 N N Corynebacteriumtuscaniae 730 AY677186 clade_102 N N Prevotella maculosa 1504AGEK01000035 clade_104 N N Prevotella oris 1513 ADDV01000091 clade_104 NN Prevotella salivae 1517 AB108826 clade_104 N N Prevotella sp. ICM551521 HQ616399 clade_104 N N Prevotella sp. oral clone AA020 1528AY005057 clade_104 N N Prevotella sp. oral clone GI032 1538 AY349396clade_104 N N Prevotella sp. oral taxon G70 1558 GU432179 clade_104 N NPrevotella corporis 1491 L16465 clade_105 N N Bacteroides sp. 4_1_36 312ACTC01000133 clade_110 N N Bacteroides sp. AR20 315 AF139524 clade_110 NN Bacteroides sp. D20 319 ACPT01000052 clade_110 N N Bacteroides sp. F_4322 AB470322 clade_110 N N Bacteroides uniformis 329 AB050110 clade_110N N Prevotella nanceiensis 1510 JN867228 clade_127 N N Prevotella sp.oral taxon 299 1548 ACWZ01000026 clade_127 N N Prevotella bergensis 1485ACKS01000100 clade_128 N N Prevotella buccalis 1489 JN867261 clade_129 NN Prevotella timonensis 1564 ADEF01000012 clade_129 N N Prevotellaoralis 1512 AEPE01000021 clade_130 N N Prevotella sp. SEQ072 1525JN867238 clade_130 N N Leuconostoc carnosum 1177 NR_040811 clade_135 N NLeuconostoc gasicomitatum 1179 FN822744 clade_135 N N Leuconostoc inhae1180 NR_025204 clade_135 N N Leuconostoc kimchii 1181 NR_075014clade_135 N N Edwardsiella tarda 777 CP002154 clade_139 N N Photorhabdusasymbiotica 1466 Z76752 clade_139 N N Psychrobacter arcticus 1607CP000082 clade_141 N N Psychrobacter cibarius 1608 HQ698586 clade_141 NN Psychrobacter cryohalolentis 1609 CP000323 clade_141 N N Psychrobacterfaecalis 1610 HQ698566 clade_141 N N Psychrobacter nivimaris 1611HQ698587 clade_141 N N Psychrobacter pulmonis 1612 HQ698582 clade_141 NN Pseudomonas aeruginosa 1592 AABQ07000001 clade_154 N N Pseudomonas sp.2_1_26 1600 ACWU01000257 clade_154 N N Corynebacterium confusum 691Y15886 clade_158 N N Corynebacterium propinquum 712 NR_037038 clade_158N N Corynebacterium pseudodiphtheriticum 713 X84258 clade_158 N NBartonella bacilliformis 338 NC_008783 clade_159 N N Bartonella grahamii339 CP001562 clade_159 N N Bartonella henselae 340 NC_005956 clade_159 NN Bartonella quintana 341 BX897700 clade_159 N N Bartonella tamiae 342EF672728 clade_159 N N Bartonella washoensis 343 FJ719017 clade_159 N NBrucella abortus 430 ACBJ01000075 clade_159 N Category-B Brucella canis431 NR_044652 clade_159 N Category-B Brucella ceti 432 ACJD01000006clade_159 N Category-B Brucella melitensis 433 AE009462 clade_159 NCategory-B Brucella microti 434 NR_042549 clade_159 N Category-BBrucella ovis 435 NC_009504 clade_159 N Category-B Brucella sp. 83_13436 ACBQ01000040 clade_159 N Category-B Brucella sp. BO1 437 EU053207clade_159 N Category-B Brucella suis 438 ACBK01000034 clade_159 NCategory-B Ochrobactrum anthropi 1360 NC_009667 clade_159 N NOchrobactrum intermedium 1361 ACQA01000001 clade_159 N N Ochrobactrumpseudintermedium 1362 DQ365921 clade_159 N N Prevotella genomosp. C21496 AY278625 clade_164 N N Prevotella multisaccharivorax 1509AFJE01000016 clade_164 N N Prevotella sp. oral clone IDR_CEC_0055 1543AY550997 clade_164 N N Prevotella sp. oral taxon 292 1547 GQ422735clade_164 N N Prevotella sp. oral taxon 300 1549 GU409549 clade_164 N NPrevotella marshii 1505 AEEI01000070 clade_166 N N Prevotella sp. oralclone IK053 1544 AY349401 clade_166 N N Prevotella sp. oral taxon 7811554 GQ422744 clade_166 N N Prevotella stercorea 1562 AB244774 clade_166N N Prevotella brevis 1487 NR_041954 clade_167 N N Prevotella ruminicola1516 CP002006 clade_167 N N Prevotella sp. sp24 1560 AB003384 clade_167N N Prevotella sp. sp34 1561 AB003385 clade_167 N N Prevotella albensis1483 NR_025300 clade_168 N N Prevotella copri 1490 ACBX02000014clade_168 N N Prevotella oulorum 1514 L16472 clade_168 N N Prevotellasp. BI_42 1518 AJ581354 clade_168 N N Prevotella sp. oral clone P4PB_83P2 1546 AY207050 clade_168 N N Prevotella sp. oral taxon G60 1557GU432133 clade_168 N N Prevotella amnii 1484 AB547670 clade_169 N NBacteroides caccae 268 EU136686 clade_170 N N Bacteroides finegoldii 277AB222699 clade_170 N N Bacteroides intestinalis 283 ABJL02000006clade_171 N N Bacteroides sp. XB44A 326 AM230649 clade_171 N NBifidobacteriaceae genomosp. C1 345 AY278612 clade_172 N NBifidobacterium adolescentis 346 AAXD02000018 clade_172 N NBifidobacterium angulatum 347 ABYS02000004 clade_172 N N Bifidobacteriumanimalis 348 CP001606 clade_172 N N Bifidobacterium breve 350 CP002743clade_172 N N Bifidobacterium catenulatum 351 ABXY01000019 clade_172 N NBifidobacterium dentium 352 CP001750 clade_172 N OP Bifidobacteriumgallicum 353 ABXB03000004 clade_172 N N Bifidobacterium infantis 354AY151398 clade_172 N N Bifidobacterium kashiwanohense 355 AB491757clade_172 N N Bifidobacterium longum 356 ABQQ01000041 clade_172 N NBifidobacterium pseudocatenulatum 357 ABXX02000002 clade_172 N NBifidobacterium pseudolongum 358 NR_043442 clade_172 N N Bifidobacteriumscardovii 359 AJ307005 clade_172 N N Bifidobacterium sp. HM2 360AB425276 clade_172 N N Bifidobacterium sp. HMLN12 361 JF519685 clade_172N N Bifidobacterium sp. M45 362 HM626176 clade_172 N N Bifidobacteriumsp. MSX5B 363 HQ616382 clade_172 N N Bifidobacterium sp. TM_7 364AB218972 clade_172 N N Bifidobacterium thermophilum 365 DQ340557clade_172 N N Leuconostoc citreum 1178 AM157444 clade_175 N NLeuconostoc lactis 1182 NR_040823 clade_175 N N Alicyclobacillusacidoterrestris 123 NR_040844 clade_179 N N Alicyclobacilluscycloheptanicus 125 NR_024754 clade_179 N N Acinetobacter baumannii 27ACYQ01000014 clade_181 N N Acinetobacter calcoaceticus 28 AM157426clade_181 N N Acinetobacter genomosp. C1 29 AY278636 clade_181 N NAcinetobacter haemolyticus 30 ADMT01000017 clade_181 N N Acinetobacterjohnsonii 31 ACPL01000162 clade_181 N N Acinetobacter junii 32ACPM01000135 clade_181 N N Acinetobacter lwoffii 33 ACPN01000204clade_181 N N Acinetobacter parvus 34 AIEB01000124 clade_181 N NAcinetobacter schindleri 36 NR_025412 clade_181 N N Acinetobacter sp.56A1 37 GQ178049 clade_181 N N Acinetobacter sp. CIP 101934 38 JQ638573clade_181 N N Acinetobacter sp. CIP 102143 39 JQ638578 clade_181 N NAcinetobacter sp. M16_22 41 HM366447 clade_181 N N Acinetobacter sp.RUH2624 42 ACQF01000094 clade_181 N N Acinetobacter sp. SH024 43ADCH01000068 clade_181 N N Lactobacillus jensenii 1092 ACQD01000066clade_182 N N Alcaligenes faecalis 119 AB680368 clade_183 N NAlcaligenes sp. CO14 120 DQ643040 clade_183 N N Alcaligenes sp. S3 121HQ262549 clade_183 N N Oligella ureolytica 1366 NR_041998 clade_183 N NOligella urethralis 1367 NR_041753 clade_183 N N Eikenella corrodens 784ACEA01000028 clade_185 N N Kingella denitrificans 1019 AEWV01000047clade_185 N N Kingella genomosp. P1 oral cone MB2_C20 1020 DQ003616clade_185 N N Kingella kingae 1021 AFHS01000073 clade_185 N N Kingellaoralis 1022 ACJW02000005 clade_185 N N Kingella sp. oral clone ID0591023 AY349381 clade_185 N N Neisseria elongata 1330 ADBF01000003clade_185 N N Neisseria genomosp. P2 oral clone MB5_P15 1332 DQ003630clade_185 N N Neisseria sp. oral clone JC012 1345 AY349388 clade_185 N NNeisseria sp. SMC_A9199 1342 FJ763637 clade_185 N N Simonsiella muelleri1731 ADCY01000105 clade_185 N N Corynebacterium glucuronolyticum 700ABYP01000081 clade_193 N N Corynebacterium pyruviciproducens 716FJ185225 clade_193 N N Rothia aeria 1649 DQ673320 clade_194 N N Rothiadentocariosa 1650 ADDW01000024 clade_194 N N Rothia sp. oral taxon 1881653 GU470892 clade_194 N N Corynebacterium accolens 681 ACGD01000048clade_195 N N Corynebacterium macginleyi 707 AB359393 clade_195 N NCorynebacterium pseudogenitalium 714 ABYQ01000237 clade_195 N NCorynebacterium tuberculostearicum 729 ACVP01000009 clade_195 N NLactobacillus casei 1074 CP000423 clade_198 N N Lactobacillus paracasei1106 ABQV01000067 clade_198 N N Lactobacillus zeae 1143 NR_037122clade_198 N N Prevotella dentalis 1492 AB547678 clade_205 N N Prevotellasp. oral clone ASCG10 1529 AY923148 clade_206 N N Prevotella sp. oralclone HF050 1541 AY349399 clade_206 N N Prevotella sp. oral clone ID0191542 AY349400 clade_206 N N Prevotella sp. oral clone IK062 1545AY349402 clade_206 N N Prevotella genomosp. P9 oral clone MB7_G16 1499DQ003633 clade_207 N N Prevotella sp. oral clone AU069 1531 AY005062clade_207 N N Prevotella sp. oral clone CY006 1532 AY005063 clade_207 NN Prevotella sp. oral clone FL019 1534 AY349392 clade_207 N NActinomyces genomosp. C1 56 AY278610 clade_212 N N Actinomyces genomosp.C2 57 AY278611 clade_212 N N Actinomyces genomosp. P1 oral clone MB6_C0358 DQ003632 clade_212 N N Actinomyces georgiae 59 GU561319 clade_212 N NActinomyces israelii 60 AF479270 clade_212 N N Actinomyces massiliensis61 AB545934 clade_212 N N Actinomyces meyeri 62 GU561321 clade_212 N NActinomyces odontolyticus 66 ACYT01000123 clade_212 N N Actinomycesorihominis 68 AJ575186 clade_212 N N Actinomyces sp. CCUG 37290 71AJ234058 clade_212 N N Actinomyces sp. ICM34 75 HQ616391 clade_212 N NActinomyces sp. ICM41 76 HQ616392 clade_212 N N Actinomyces sp. ICM47 77HQ616395 clade_212 N N Actinomyces sp. ICM54 78 HQ616398 clade_212 N NActinomyces sp. oral clone IP081 87 AY349366 clade_212 N N Actinomycessp. oral taxon 178 91 AEUH01000060 clade_212 N N Actinomyces sp. oraltaxon 180 92 AEPP01000041 clade_212 N N Actinomyces sp. TeJ5 80 GU561315clade_212 N N Haematobacter sp. BC14248 968 GU396991 clade_213 N NParacoccus denitrificans 1424 CP000490 clade_213 N N Paracoccus marcusii1425 NR_044922 clade_213 N N Grimontia hollisae 967 ADAQ01000013clade_216 N N Shewanella putrefaciens 1723 CP002457 clade_216 N N Afipiagenomosp. 4 111 EU117385 clade_217 N N Rhodopseudomonas palustris 1626CP000301 clade_217 N N Methylobacterium extorquens 1223 NC_010172clade_218 N N Methylobacterium podarium 1224 AY468363 clade_218 N NMethylobacterium radiotolerans 1225 GU294320 clade_218 N NMethylobacterium sp. 1sub 1226 AY468371 clade_218 N N Methylobacteriumsp. MM4 1227 AY468370 clade_218 N N Achromobacter denitrificans 18NR_042021 clade_224 N N Achromobacter piechaudii 19 ADMS01000149clade_224 N N Achromobacter xylosoxidans 20 ACRC01000072 clade_224 N NBordetella bronchiseptica 384 NR_025949 clade_224 N OP Bordetellaholmesii 385 AB683187 clade_224 N OP Bordetella parapertussis 386NR_025950 clade_224 N OP Bordetella pertussis 387 BX640418 clade_224 NOP Microbacterium chocolatum 1230 NR_037045 clade_225 N N Microbacteriumflavescens 1231 EU714363 clade_225 N N Microbacterium lacticum 1233EU714351 clade_225 N N Microbacterium oleivorans 1234 EU714381 clade_225N N Microbacterium oxydans 1235 EU714348 clade_225 N N Microbacteriumparaoxydans 1236 AJ491806 clade_225 N N Microbacterium phyllosphaerae1237 EU714359 clade_225 N N Microbacterium schleiferi 1238 NR_044936clade_225 N N Microbacterium sp. 768 1239 EU714378 clade_225 N NMicrobacterium sp. oral strain C24KA 1240 AF287752 clade_225 N NMicrobacterium testaceum 1241 EU714365 clade_225 N N Corynebacteriumatypicum 686 NR_025540 clade_229 N N Corynebacterium mastitidis 708AB359395 clade_229 N N Corynebacterium sp. NML 97_0186 725 GU238411clade_229 N N Mycobacterium elephantis 1275 AF385898 clade_237 N OPMycobacterium paraterrae 1288 EU919229 clade_237 N OP Mycobacteriumphlei 1289 GU142920 clade_237 N OP Mycobacterium sp. 1776 1293 EU703152clade_237 N N Mycobacterium sp. 1781 1294 EU703147 clade_237 N NMycobacterium sp. AQ1GA4 1297 HM210417 clade_237 N N Mycobacterium sp.GN_10546 1299 FJ497243 clade_237 N N Mycobacterium sp. GN_10827 1300FJ497247 clade_237 N N Mycobacterium sp. GN_11124 1301 FJ652846clade_237 N N Mycobacterium sp. GN_9188 1302 FJ497240 clade_237 N NMycobacterium sp. GR_2007_210 1303 FJ555538 clade_237 N N Anoxybacilluscontaminans 172 NR_029006 clade_238 N N Bacillus aeolius 195 NR_025557clade_238 N N Brevibacterium frigoritolerans 422 NR_042639 clade_238 N NGeobacillus sp. E263 934 DQ647387 clade_238 N N Geobacillus sp. WCH70935 CP001638 clade_238 N N Geobacillus thermocatenulatus 937 NR_043020clade_238 N N Geobacillus thermoleovorans 940 NR_074931 clade_238 N NLysinibacillus fusiformis 1192 FN397522 clade_238 N N Planomicrobiumkoreense 1468 NR_025011 clade_238 N N Sporosarcina newyorkensis 1754AFPZ01000142 clade_238 N N Sporosarcina sp. 2681 1755 GU994081 clade_238N N Ureibacillus composti 1968 NR_043746 clade_238 N N Ureibacillussuwonensis 1969 NR_043232 clade_238 N N Ureibacillus terrenus 1970NR_025394 clade_238 N N Ureibacillus thermophilus 1971 NR_043747clade_238 N N Ureibacillus thermosphaericus 1972 NR_040961 clade_238 N NPrevotella micans 1507 AGWK01000061 clade_239 N N Prevotella sp. oralclone DA058 1533 AY005065 clade_239 N N Prevotella sp. SEQ053 1523JN867222 clade_239 N N Treponema socranskii 1937 NR_024868 clade_240 NOP Treponema sp. 6:H:D15A_4 1938 AY005083 clade_240 N N Treponema sp.oral taxon 265 1953 GU408850 clade_240 N N Treponema sp. oral taxon G851958 GU432215 clade_240 N N Porphyromonas endodontalis 1472 ACNN01000021clade_241 N N Porphyromonas sp. oral clone BB134 1478 AY005068 clade_241N N Porphyromonas sp. oral clone F016 1479 AY005069 clade_241 N NPorphyromonas sp. oral clone P2PB_52 P1 1480 AY207054 clade_241 N NPorphyromonas sp. oral clone P4GB_100 P2 1481 AY207057 clade_241 N NAcidovorax sp. 98_63833 26 AY258065 clade_245 N N Comamonadaceaebacterium NML000135 663 JN585335 clade_245 N N Comamonadaceae bacteriumNML790751 664 JN585331 clade_245 N N Comamonadaceae bacterium NML910035665 JN585332 clade_245 N N Comamonadaceae bacterium NML910036 666JN585333 clade_245 N N Comamonas sp. NSP5 668 AB076850 clade_245 N NDelftia acidovorans 748 CP000884 clade_245 N N Xenophilus aerolatus 2018JN585329 clade_245 N N Oribacterium sp. oral taxon 078 1380 ACIQ02000009clade_246 N N Oribacterium sp. oral taxon 102 1381 GQ422713 clade_246 NN Weissella cibaria 2007 NR_036924 clade_247 N N Weissella confusa 2008NR_040816 clade_247 N N Weissella hellenica 2009 AB680902 clade_247 N NWeissella kandleri 2010 NR_044659 clade_247 N N Weissella koreensis 2011NR_075058 clade_247 N N Weissella paramesenteroides 2012 ACKU01000017clade_247 N N Weissella sp. KLDS 7.0701 2013 EU600924 clade_247 N NMobiluncus curtisii 1251 AEPZ01000013 clade_249 N N Enhydrobacteraerosaccus 785 ACYI01000081 clade_256 N N Moraxella osloensis 1262JN175341 clade_256 N N Moraxella sp. GM2 1264 JF837191 clade_256 N NBrevibacterium casei 420 JF951998 clade_257 N N Brevibacteriumepidermidis 421 NR_029262 clade_257 N N Brevibacterium sanguinis 426NR_028016 clade_257 N N Brevibacterium sp. H15 427 AB177640 clade_257 NN Acinetobacter radioresistens 35 ACVR01000010 clade_261 N NLactobacillus alimentarius 1068 NR_044701 clade_263 N N Lactobacillusfarciminis 1082 NR_044707 clade_263 N N Lactobacillus kimchii 1097NR_025045 clade_263 N N Lactobacillus nodensis 1101 NR_041629 clade_263N N Lactobacillus tucceti 1138 NR_042194 clade_263 N N Pseudomonasmendocina 1595 AAUL01000021 clade_265 N N Pseudomonas pseudoalcaligenes1598 NR_037000 clade_265 N N Pseudomonas sp. NP522b 1602 EU723211clade_265 N N Pseudomonas stutzeri 1603 AM905854 clade_265 N NPaenibacillus barcinonensis 1390 NR_042272 clade_270 N N Paenibacillusbarengoltzii 1391 NR_042756 clade_270 N N Paenibacillus chibensis 1392NR_040885 clade_270 N N Paenibacillus cookii 1393 NR_025372 clade_270 NN Paenibacillus durus 1394 NR_037017 clade_270 N N Paenibacillusglucanolyticus 1395 D78470 clade_270 N N Paenibacillus lactis 1396NR_025739 clade_270 N N Paenibacillus pabuli 1398 NR_040853 clade_270 NN Paenibacillus popilliae 1400 NR_040888 clade_270 N N Paenibacillus sp.CIP 101062 1401 HM212646 clade_270 N N Paenibacillus sp. JC66 1404JF824808 clade_270 N N Paenibacillus sp. R_27413 1405 HE586333 clade_270N N Paenibacillus sp. R_27422 1406 HE586338 clade_270 N N Paenibacillustimonensis 1408 NR_042844 clade_270 N N Rothia mucilaginosa 1651ACVO01000020 clade_271 N N Rothia nasimurium 1652 NR_025310 clade_271 NN Prevotella sp. oral taxon 302 1550 ACZK01000043 clade_280 N NPrevotella sp. oral taxon F68 1556 HM099652 clade_280 N N Prevotellatannerae 1563 ACIJ02000018 clade_280 N N Prevotellaceae bacterium P4P_62P1 1566 AY207061 clade_280 N N Porphyromonas asaccharolytica 1471AENO01000048 clade_281 N N Porphyromonas gingivalis 1473 AE015924clade_281 N N Porphyromonas macacae 1475 NR_025908 clade_281 N NPorphyromonas sp. UQD 301 1477 EU012301 clade_281 N N Porphyromonasuenonis 1482 ACLR01000152 clade_281 N N Leptotrichia buccalis 1165CP001685 clade_282 N N Leptotrichia hofstadii 1168 ACVB02000032clade_282 N N Leptotrichia sp. oral clone HE012 1173 AY349386 clade_282N N Leptotrichia sp. oral taxon 223 1176 GU408547 clade_282 N NBacteroides fluxus 278 AFBN01000029 clade_285 N N Bacteroides helcogenes281 CP002352 clade_285 N N Parabacteroides johnsonii 1419 ABYH01000014clade_286 N N Parabacteroides merdae 1420 EU136685 clade_286 N NTreponema denticola 1926 ADEC01000002 clade_288 N OP Treponema genomosp.P5 oral clone MB3_P23 1929 DQ003624 clade_288 N N Treponema putidum 1935AJ543428 clade_288 N OP Treponema sp. oral clone P2PB_53 P3 1942AY207055 clade_288 N N Treponema sp. oral taxon 247 1949 GU408748clade_288 N N Treponema sp. oral taxon 250 1950 GU408776 clade_288 N NTreponema sp. oral taxon 251 1951 GU408781 clade_288 N N Anaerococcushydrogenalis 144 ABXA01000039 clade_289 N N Anaerococcus sp. 8404299 148HM587318 clade_289 N N Anaerococcus sp. gpac215 156 AM176540 clade_289 NN Anaerococcus vaginalis 158 ACXU01000016 clade_289 N NPropionibacterium acidipropionici 1569 NC_019395 clade_290 N NPropionibacterium avidum 1571 AJ003055 clade_290 N N Propionibacteriumgranulosum 1573 FJ785716 clade_290 N N Propionibacterium jensenii 1574NR_042269 clade_290 N N Propionibacterium propionicum 1575 NR_025277clade_290 N N Propionibacterium sp. H456 1577 AB177643 clade_290 N NPropionibacterium thoenii 1581 NR_042270 clade_290 N N Bifidobacteriumbifidum 349 ABQP01000027 clade_293 N N Leuconostoc mesenteroides 1183ACKV01000113 clade_295 N N Leuconostoc pseudomesenteroides 1184NR_040814 clade_295 N N Johnsonella ignava 1016 X87152 clade_298 N NPropionibacterium acnes 1570 ADJM01000010 clade_299 N NPropionibacterium sp. 434_HC2 1576 AFIL01000035 clade_299 N NPropionibacterium sp. LG 1578 AY354921 clade_299 N N Propionibacteriumsp. S555a 1579 AB264622 clade_299 N N Alicyclobacillus sp. CCUG 53762128 HE613268 clade_301 N N Actinomyces cardiffensis 53 GU470888clade_303 N N Actinomyces funkei 55 HQ906497 clade_303 N N Actinomycessp. HKU31 74 HQ335393 clade_303 N N Actinomyces sp. oral taxon C55 94HM099646 clade_303 N N Kerstersia gyiorum 1018 NR_025669 clade_307 N NPigmentiphaga daeguensis 1467 JN585327 clade_307 N N Aeromonasallosaccharophila 104 S39232 clade_308 N N Aeromonas enteropelogenes 105X71121 clade_308 N N Aeromonas hydrophila 106 NC_008570 clade_308 N NAeromonas jandaei 107 X60413 clade_308 N N Aeromonas salmonicida 108NC_009348 clade_308 N N Aeromonas trota 109 X60415 clade_308 N NAeromonas veronii 110 NR_044845 clade_308 N N Marvinbryantiaformatexigens 1196 AJ505973 clade_309 N N Rhodobacter sp. oral taxon C301620 HM099648 clade_310 N N Rhodobacter sphaeroides 1621 CP000144clade_310 N N Lactobacillus antri 1071 ACLL01000037 clade_313 N NLactobacillus coleohominis 1076 ACOH01000030 clade_313 N N Lactobacillusfermentum 1083 CP002033 clade_313 N N Lactobacillus gastricus 1085AICN01000060 clade_313 N N Lactobacillus mucosae 1099 FR693800 clade_313N N Lactobacillus oris 1103 AEKL01000077 clade_313 N N Lactobacilluspontis 1111 HM218420 clade_313 N N Lactobacillus reuteri 1112ACGW02000012 clade_313 N N Lactobacillus sp. KLDS 1.0707 1127 EU600911clade_313 N N Lactobacillus sp. KLDS 1.0709 1128 EU600913 clade_313 N NLactobacillus sp. KLDS 1.0711 1129 EU600915 clade_313 N N Lactobacillussp. KLDS 1.0713 1131 EU600917 clade_313 N N Lactobacillus sp. KLDS1.0716 1132 EU600921 clade_313 N N Lactobacillus sp. KLDS 1.0718 1133EU600922 clade_313 N N Lactobacillus sp. oral taxon 052 1137 GQ422710clade_313 N N Lactobacillus vaginalis 1140 ACGV01000168 clade_313 N NBrevibacterium aurantiacum 419 NR_044854 clade_314 N N Brevibacteriumlinens 423 AJ315491 clade_314 N N Lactobacillus pentosus 1108 JN813103clade_315 N N Lactobacillus plantarum 1110 ACGZ02000033 clade_315 N NLactobacillus sp. KLDS 1.0702 1123 EU600906 clade_315 N N Lactobacillussp. KLDS 1.0703 1124 EU600907 clade_315 N N Lactobacillus sp. KLDS1.0704 1125 EU600908 clade_315 N N Lactobacillus sp. KLDS 1.0705 1126EU600909 clade_315 N N Agrobacterium radiobacter 115 CP000628 clade_316N N Agrobacterium tumefaciens 116 AJ389893 clade_316 N N Corynebacteriumargentoratense 685 EF463055 clade_317 N N Corynebacterium diphtheriae693 NC_002935 clade_317 N OP Corynebacterium pseudotuberculosis 715NR_037070 clade_317 N N Corynebacterium renale 717 NR_037069 clade_317 NN Corynebacterium ulcerans 731 NR_074467 clade_317 N N Aurantimonascoralicida 191 AY065627 clade_318 N N Aureimonas altamirensis 192FN658986 clade_318 N N Lactobacillus acidipiscis 1066 NR_024718clade_320 N N Lactobacillus salivarius 1117 AEBA01000145 clade_320 N NLactobacillus sp. KLDS 1.0719 1134 EU600923 clade_320 N N Lactobacillusbuchneri 1073 ACGH01000101 clade_321 N N Lactobacillus genomosp. C1 1086AY278619 clade_321 N N Lactobacillus genomosp. C2 1087 AY278620clade_321 N N Lactobacillus hilgardii 1089 ACGP01000200 clade_321 N NLactobacillus kefiri 1096 NR_042230 clade_321 N N Lactobacillusparabuchneri 1105 NR_041294 clade_321 N N Lactobacillus parakefiri 1107NR_029039 clade_321 N N Lactobacillus curvatus 1079 NR_042437 clade_322N N Lactobacillus sakei 1116 DQ989236 clade_322 N N Aneurinibacillusaneurinilyticus 167 AB101592 clade_323 N N Aneurinibacillus danicus 168NR_028657 clade_323 N N Aneurinibacillus migulanus 169 NR_036799clade_323 N N Aneurinibacillus terranovensis 170 NR_042271 clade_323 N NStaphylococcus aureus 1757 CP002643 clade_325 N Category-BStaphylococcus auricularis 1758 JQ624774 clade_325 N N Staphylococcuscapitis 1759 ACFR01000029 clade_325 N N Staphylococcus caprae 1760ACRH01000033 clade_325 N N Staphylococcus carnosus 1761 NR_075003clade_325 N N Staphylococcus cohnii 1762 JN175375 clade_325 N NStaphylococcus condimenti 1763 NR_029345 clade_325 N N Staphylococcusepidermidis 1764 ACHE01000056 clade_325 N N Staphylococcus equorum 1765NR_027520 clade_325 N N Staphylococcus haemolyticus 1767 NC_007168clade_325 N N Staphylococcus hominis 1768 AM157418 clade_325 N NStaphylococcus lugdunensis 1769 AEQA01000024 clade_325 N NStaphylococcus pasteuri 1770 FJ189773 clade_325 N N Staphylococcuspseudintermedius 1771 CP002439 clade_325 N N Staphylococcussaccharolyticus 1772 NR_029158 clade_325 N N Staphylococcussaprophyticus 1773 NC_007350 clade_325 N N Staphylococcus sp. clonebottae7 1777 AF467424 clade_325 N N Staphylococcus sp. H292 1775AB177642 clade_325 N N Staphylococcus sp. H780 1776 AB177644 clade_325 NN Staphylococcus succinus 1778 NR_028667 clade_325 N N Staphylococcuswarneri 1780 ACPZ01000009 clade_325 N N Staphylococcus xylosus 1781AY395016 clade_325 N N Cardiobacterium hominis 490 ACKY01000036clade_326 N N Cardiobacterium valvarum 491 NR_028847 clade_326 N NPseudomonas fluorescens 1593 AY622220 clade_326 N N Pseudomonasgessardii 1594 FJ943496 clade_326 N N Pseudomonas monteilii 1596NR_024910 clade_326 N N Pseudomonas poae 1597 GU188951 clade_326 N NPseudomonas putida 1599 AF094741 clade_326 N N Pseudomonas sp. G12291601 DQ910482 clade_326 N N Pseudomonas tolaasii 1604 AF320988 clade_326N N Pseudomonas viridiflava 1605 NR_042764 clade_326 N N Listeria grayi1185 ACCR02000003 clade_328 N OP Listeria innocua 1186 JF967625clade_328 N N Listeria ivanovii 1187 X56151 clade_328 N N Listeriamonocytogenes 1188 CP002003 clade_328 N Category-B Listeria welshimeri1189 AM263198 clade_328 N OP Capnocytophaga sp. oral clone ASCH05 484AY923149 clade_333 N N Capnocytophaga sputigena 489 ABZV01000054clade_333 N N Leptotrichia genomosp. C1 1166 AY278621 clade_334 N NLeptotrichia shahii 1169 AY029806 clade_334 N N Leptotrichia sp.neutropenicPatient 1170 AF189244 clade_334 N N Leptotrichia sp. oralclone GT018 1171 AY349384 clade_334 N N Leptotrichia sp. oral cloneGT020 1172 AY349385 clade_334 N N Bacteroides sp. 20_3 296 ACRQ01000064clade_335 N N Bacteroides sp. 3_1_19 307 ADCJ01000062 clade_335 N NBacteroides sp. 3_2_5 311 ACIB01000079 clade_335 N N Parabacteroidesdistasonis 1416 CP000140 clade_335 N N Parabacteroides goldsteinii 1417AY974070 clade_335 N N Parabacteroides gordonii 1418 AB470344 clade_335N N Parabacteroides sp. D13 1421 ACPW01000017 clade_335 N NCapnocytophaga genomosp. C1 477 AY278613 clade_336 N N Capnocytophagaochracea 480 AEOH01000054 clade_336 N N Capnocytophaga sp. GEJ8 481GU561335 clade_336 N N Capnocytophaga sp. oral strain A47ROY 486AY005077 clade_336 N N Capnocytophaga sp. S1b 482 U42009 clade_336 N NParaprevotella clara 1426 AFFY01000068 clade_336 N N Bacteroidesheparinolyticus 282 JN867284 clade_338 N N Prevotella heparinolytica1500 GQ422742 clade_338 N N Treponema genomosp. P4 oral clone MB2_G191928 DQ003618 clade_339 N N Treponema genomosp. P6 oral clone MB4_G111930 DQ003625 clade_339 N N Treponema sp. oral taxon 254 1952 GU408803clade_339 N N Treponema sp. oral taxon 508 1956 GU413616 clade_339 N NTreponema sp. oral taxon 518 1957 GU413640 clade_339 N N Chlamydiamuridarum 502 AE002160 clade_341 N OP Chlamydia trachomatis 504 U68443clade_341 N OP Chlamydia psittaci 503 NR_036864 clade_342 N Category-BChlamydophila pneumoniae 509 NC_002179 clade_342 N OP Chlamydophilapsittaci 510 D85712 clade_342 N OP Anaerococcus octavius 146 NR_026360clade_343 N N Anaerococcus sp. 8405254 149 HM587319 clade_343 N NAnaerococcus sp. 9401487 150 HM587322 clade_343 N N Anaerococcus sp.9403502 151 HM587325 clade_343 N N Gardnerella vaginalis 923 CP001849clade_344 N N Campylobacter lari 466 CP000932 clade_346 N OPAnaerobiospirillum succiniciproducens 142 NR_026075 clade_347 N NAnaerobiospirillum thomasii 143 AJ420985 clade_347 N N Ruminobacteramylophilus 1654 NR_026450 clade_347 N N Succinatimonas hippei 1897AEVO01000027 clade_347 N N Actinomyces europaeus 54 NR_026363 clade_348N N Actinomyces sp. oral clone GU009 82 AY349361 clade_348 N N Moraxellacatarrhalis 1260 CP002005 clade_349 N N Moraxella lincolnii 1261FR822735 clade_349 N N Moraxella sp. 16285 1263 JF682466 clade_349 N NPsychrobacter sp. 13983 1613 HM212668 clade_349 N N Actinobaculummassiliae 49 AF487679 clade_350 N N Actinobaculum schaalii 50 AY957507clade_350 N N Actinobaculum sp. BM#101342 51 AY282578 clade_350 N NActinobaculum sp. P2P_19 P1 52 AY207066 clade_350 N N Actinomyces sp.oral clone IO076 84 AY349363 clade_350 N N Actinomyces sp. oral taxon848 93 ACUY01000072 clade_350 N N Actinomyces neuii 65 X71862 clade_352N N Mobiluncus mulieris 1252 ACKW01000035 clade_352 N N Blastomonasnatatoria 372 NR_040824 clade_356 N N Novosphingobium aromaticivorans1357 AAAV03000008 clade_356 N N Sphingomonas sp. oral clone FI012 1745AY349411 clade_356 N N Sphingopyxis alaskensis 1749 CP000356 clade_356 NN Oxalobacter formigenes 1389 ACDQ01000020 clade_357 N N Veillonellaatypica 1974 AEDS01000059 clade_358 N N Veillonella dispar 1975ACIK02000021 clade_358 N N Veillonella genomosp. P1 oral clone MB5_P171976 DQ003631 clade_358 N N Veillonella parvula 1978 ADFU01000009clade_358 N N Veillonella sp. 3_1_44 1979 ADCV01000019 clade_358 N NVeillonella sp. 6_1_27 1980 ADCW01000016 clade_358 N N Veillonella sp.ACP1 1981 HQ616359 clade_358 N N Veillonella sp. AS16 1982 HQ616365clade_358 N N Veillonella sp. BS32b 1983 HQ616368 clade_358 N NVeillonella sp. ICM51a 1984 HQ616396 clade_358 N N Veillonella sp. MSA121985 HQ616381 clade_358 N N Veillonella sp. NVG 100cf 1986 EF108443clade_358 N N Veillonella sp. OK11 1987 JN695650 clade_358 N NVeillonella sp. oral clone ASCG01 1990 AY923144 clade_358 N NVeillonella sp. oral clone ASCG02 1991 AY953257 clade_358 N NVeillonella sp. oral clone OH1A 1992 AY947495 clade_358 N N Veillonellasp. oral taxon 158 1993 AENU01000007 clade_358 N N Kocuria marina 1040GQ260086 clade_365 N N Kocuria rhizophila 1042 AY030315 clade_365 N NKocuria rosea 1043 X87756 clade_365 N N Kocuria varians 1044 AF542074clade_365 N N Clostridiaceae bacterium END_2 531 EF451053 clade_368 N NMicrococcus antarcticus 1242 NR_025285 clade_371 N N Micrococcus luteus1243 NR_075062 clade_371 N N Micrococcus lylae 1244 NR_026200 clade_371N N Micrococcus sp. 185 1245 EU714334 clade_371 N N Lactobacillus brevis1072 EU194349 clade_372 N N Lactobacillus parabrevis 1104 NR_042456clade_372 N N Pediococcus acidilactici 1436 ACXB01000026 clade_372 N NPediococcus pentosaceus 1437 NR_075052 clade_372 N N Lactobacillusdextrinicus 1081 NR_036861 clade_373 N N Lactobacillus perolens 1109NR_029360 clade_373 N N Lactobacillus rhamnosus 1113 ABWJ01000068clade_373 N N Lactobacillus saniviri 1118 AB602569 clade_373 N NLactobacillus sp. BT6 1121 HQ616370 clade_373 N N Mycobacteriummageritense 1282 FR798914 clade_374 N OP Mycobacterium neoaurum 1286AF268445 clade_374 N OP Mycobacterium smegmatis 1291 CP000480 clade_374N OP Mycobacterium sp. HE5 1304 AJ012738 clade_374 N N Dysgonomonasgadei 775 ADLV01000001 clade_377 N N Dysgonomonas mossii 776ADLW01000023 clade_377 N N Porphyromonas levii 1474 NR_025907 clade_377N N Porphyromonas somerae 1476 AB547667 clade_377 N N Bacteroidesbarnesiae 267 NR_041446 clade_378 N N Bacteroides coprocola 272ABIY02000050 clade_378 N N Bacteroides coprophilus 273 ACBW01000012clade_378 N N Bacteroides dorei 274 ABWZ01000093 clade_378 N NBacteroides massiliensis 284 AB200226 clade_378 N N Bacteroides plebeius289 AB200218 clade_378 N N Bacteroides sp. 3_1_33FAA 309 ACPS01000085clade_378 N N Bacteroides sp. 3_1_40A 310 ACRT01000136 clade_378 N NBacteroides sp. 4_3_47FAA 313 ACDR02000029 clade_378 N N Bacteroides sp.9_1_42FAA 314 ACAA01000096 clade_378 N N Bacteroides sp. NB_8 323AB117565 clade_378 N N Bacteroides vulgatus 331 CP000139 clade_378 N NBacteroides ovatus 287 ACWH01000036 clade_38 N N Bacteroides sp. 1_1_30294 ADCL01000128 clade_38 N N Bacteroides sp. 2_1_22 297 ACPQ01000117clade_38 N N Bacteroides sp. 2_2_4 299 ABZZ01000168 clade_38 N NBacteroides sp. 3_1_23 308 ACRS01000081 clade_38 N N Bacteroides sp. D1318 ACAB02000030 clade_38 N N Bacteroides sp. D2 321 ACGA01000077clade_38 N N Bacteroides sp. D22 320 ADCK01000151 clade_38 N NBacteroides xylanisolvens 332 ADKP01000087 clade_38 N N Treponemalecithinolyticum 1931 NR_026247 clade_380 N OP Treponema parvum 1933AF302937 clade_380 N OP Treponema sp. oral clone JU025 1940 AY349417clade_380 N N Treponema sp. oral taxon 270 1954 GQ422733 clade_380 N NParascardovia denticolens 1428 ADEB01000020 clade_381 N N Scardoviainopinata 1688 AB029087 clade_381 N N Scardovia wiggsiae 1689 AY278626clade_381 N N Clostridiales bacterium 9400853 533 HM587320 clade_384 N NMogibacterium diversum 1254 NR_027191 clade_384 N N Mogibacteriumneglectum 1255 NR_027203 clade_384 N N Mogibacterium pumilum 1256NR_028608 clade_384 N N Mogibacterium timidum 1257 Z36296 clade_384 N NBorrelia burgdorferi 389 ABGI01000001 clade_386 N OP Borrelia garinii392 ABJV01000001 clade_386 N OP Borrelia sp. NE49 397 AJ224142 clade_386N OP Caldimonas manganoxidans 457 NR_040787 clade_387 N N Comamonadaceaebacterium oral taxon F47 667 HM099651 clade_387 N N Lautropia mirabilis1149 AEQP01000026 clade_387 N N Lautropia sp. oral clone AP009 1150AY005030 clade_387 N N Peptoniphilus asaccharolyticus 1441 D14145clade_389 N N Peptoniphilus duerdenii 1442 EU526290 clade_389 N NPeptoniphilus harei 1443 NR_026358 clade_389 N N Peptoniphilus indolicus1444 AY153431 clade_389 N N Peptoniphilus lacrimalis 1446 ADDO01000050clade_389 N N Peptoniphilus sp. gpac077 1450 AM176527 clade_389 N NPeptoniphilus sp. JC140 1447 JF824803 clade_389 N N Peptoniphilus sp.oral taxon 386 1452 ADCS01000031 clade_389 N N Peptoniphilus sp. oraltaxon 836 1453 AEAA01000090 clade_389 N N Peptostreptococcaceaebacterium ph1 1454 JN837495 clade_389 N N Dialister pneumosintes 765HM596297 clade_390 N N Dialister sp. oral taxon 502 767 GQ422739clade_390 N N Cupriavidus metallidurans 741 GU230889 clade_391 N NHerbaspirillum seropedicae 1001 CP002039 clade_391 N N Herbaspirillumsp. JC206 1002 JN657219 clade_391 N N Janthinobacterium sp. SY12 1015EF455530 clade_391 N N Massilia sp. CCUG 43427A 1197 FR773700 clade_391N N Ralstonia pickettii 1615 NC_010682 clade_391 N N Ralstonia sp.5_7_47FAA 1616 ACUF01000076 clade_391 N N Francisella novicida 889ABSS01000002 clade_392 N N Francisella philomiragia 890 AY928394clade_392 N N Francisella tularensis 891 ABAZ01000082 clade_392 NCategory-A Ignatzschineria indica 1009 HQ823562 clade_392 N NIgnatzschineria sp. NML 95_0260 1010 HQ823559 clade_392 N NStreptococcus mutans 1814 AP010655 clade_394 N N Lactobacillus gasseri1084 ACOZ01000018 clade_398 N N Lactobacillus hominis 1090 FR681902clade_398 N N Lactobacillus iners 1091 AEKJ01000002 clade_398 N NLactobacillus johnsonii 1093 AE017198 clade_398 N N Lactobacillussenioris 1119 AB602570 clade_398 N N Lactobacillus sp. oral clone HT0021135 AY349382 clade_398 N N Weissella beninensis 2006 EU439435 clade_398N N Sphingomonas echinoides 1744 NR_024700 clade_399 N N Sphingomonassp. oral taxon A09 1747 HM099639 clade_399 N N Sphingomonas sp. oraltaxon F71 1748 HM099645 clade_399 N N Zymomonas mobilis 2032 NR_074274clade_399 N N Arcanobacterium haemolyticum 174 NR_025347 clade_400 N NArcanobacterium pyogenes 175 GU585578 clade_400 N N Trueperella pyogenes1962 NR_044858 clade_400 N N Lactococcus garvieae 1144 AF061005clade_401 N N Lactococcus lactis 1145 CP002365 clade_401 N NBrevibacterium mcbrellneri 424 ADNU01000076 clade_402 N N Brevibacteriumpaucivorans 425 EU086796 clade_402 N N Brevibacterium sp. JC43 428JF824806 clade_402 N N Selenomonas artemidis 1692 HM596274 clade_403 N NSelenomonas sp. FOBRC9 1704 HQ616378 clade_403 N N Selenomonas sp. oraltaxon 137 1715 AENV01000007 clade_403 N N Desmospora activa 751 AM940019clade_404 N N Desmospora sp. 8437 752 AFHT01000143 clade_404 N NPaenibacillus sp. oral taxon F45 1407 HM099647 clade_404 N NCorynebacterium ammoniagenes 682 ADNS01000011 clade_405 N NCorynebacterium aurimucosum 687 ACLH01000041 clade_405 N NCorynebacterium bovis 688 AF537590 clade_405 N N Corynebacterium canis689 GQ871934 clade_405 N N Corynebacterium casei 690 NR_025101 clade_405N N Corynebacterium durum 694 Z97069 clade_405 N N Corynebacteriumefficiens 695 ACLI01000121 clade_405 N N Corynebacterium falsenii 696Y13024 clade_405 N N Corynebacterium flavescens 697 NR_037040 clade_405N N Corynebacterium glutamicum 701 BA000036 clade_405 N NCorynebacterium jeikeium 704 ACYW01000001 clade_405 N OP Corynebacteriumkroppenstedtii 705 NR_026380 clade_405 N N Corynebacteriumlipophiloflavum 706 ACHJ01000075 clade_405 N N Corynebacteriummatruchotii 709 ACSH02000003 clade_405 N N Corynebacterium minutissimum710 X82064 clade_405 N N Corynebacterium resistens 718 ADGN01000058clade_405 N N Corynebacterium simulans 720 AF537604 clade_405 N NCorynebacterium singulare 721 NR_026394 clade_405 N N Corynebacteriumsp. 1 ex sheep 722 Y13427 clade_405 N N Corynebacterium sp. NML 99_0018726 GU238413 clade_405 N N Corynebacterium striatum 727 ACGE01000001clade_405 N OP Corynebacterium urealyticum 732 X81913 clade_405 N OPCorynebacterium variabile 734 NR_025314 clade_405 N N Aerococcussanguinicola 98 AY837833 clade_407 N N Aerococcus urinae 99 CP002512clade_407 N N Aerococcus urinaeequi 100 NR_043443 clade_407 N NAerococcus viridans 101 ADNT01000041 clade_407 N N Fusobacteriumnaviforme 898 HQ223106 clade_408 N N Moryella indoligenes 1268 AF527773clade_408 N N Selenomonas genomosp. P5 1697 AY341820 clade_410 N NSelenomonas sp. oral clone IQ048 1710 AY349408 clade_410 N N Selenomonassputigena 1717 ACKP02000033 clade_410 N N Hyphomicrobium sulfonivorans1007 AY468372 clade_411 N N Methylocella silvestris 1228 NR_074237clade_411 N N Legionella pneumophila 1153 NC_002942 clade_412 N OPLactobacillus coryniformis 1077 NR_044705 clade_413 N N Arthrobacteragilis 178 NR_026198 clade_414 N N Arthrobacter arilaitensis 179NR_074608 clade_414 N N Arthrobacter bergerei 180 NR_025612 clade_414 NN Arthrobacter globiformis 181 NR_026187 clade_414 N N Arthrobacternicotianae 182 NR_026190 clade_414 N N Mycobacterium abscessus 1269AGQU01000002 clade_418 N OP Mycobacterium chelonae 1273 AB548610clade_418 N OP Bacteroides salanitronis 291 CP002530 clade_419 N NParaprevotella xylaniphila 1427 AFBR01000011 clade_419 N N Barnesiellaintestinihominis 336 AB370251 clade_420 N N Barnesiella viscericola 337NR_041508 clade_420 N N Parabacteroides sp. NS31_3 1422 JN029805clade_420 N N Porphyromonadaceae bacterium NML 060648 1470 EF184292clade_420 N N Tannerella forsythia 1913 CP003191 clade_420 N NTannerella sp. 6_1_58FAA_CT1 1914 ACWX01000068 clade_420 N N Mycoplasmaamphoriforme 1311 AY531656 clade_421 N N Mycoplasma genitalium 1317L43967 clade_421 N N Mycoplasma pneumoniae 1322 NC_000912 clade_421 N NMycoplasma penetrans 1321 NC_004432 clade_422 N N Ureaplasma parvum 1966AE002127 clade_422 N N Ureaplasma urealyticum 1967 AAYN01000002clade_422 N N Treponema genomosp. P1 1927 AY341822 clade_425 N NTreponema sp. oral taxon 228 1943 GU408580 clade_425 N N Treponema sp.oral taxon 230 1944 GU408603 clade_425 N N Treponema sp. oral taxon 2311945 GU408631 clade_425 N N Treponema sp. oral taxon 232 1946 GU408646clade_425 N N Treponema sp. oral taxon 235 1947 GU408673 clade_425 N NTreponema sp. ovine footrot 1959 AJ010951 clade_425 N N Treponemavincentii 1960 ACYH01000036 clade_425 N OP Burkholderiales bacterium1_1_47 452 ADCQ01000066 clade_432 N OP Parasutterella excrementihominis1429 AFBP01000029 clade_432 N N Parasutterella secunda 1430 AB491209clade_432 N N Sutterella morbirenis 1898 AJ832129 clade_432 N NSutterella sanguinus 1900 AJ748647 clade_432 N N Sutterella sp. YIT12072 1901 AB491210 clade_432 N N Sutterella stercoricanis 1902NR_025600 clade_432 N N Sutterella wadsworthensis 1903 ADMF01000048clade_432 N N Propionibacterium freudenreichii 1572 NR_036972 clade_433N N Propionibacterium sp. oral taxon 192 1580 GQ422728 clade_433 N NTessaracoccus sp. oral taxon F04 1917 HM099640 clade_433 N NPeptoniphilus ivorii 1445 Y07840 clade_434 N N Peptoniphilus sp. gpac0071448 AM176517 clade_434 N N Peptoniphilus sp. gpac018A 1449 AM176519clade_434 N N Peptoniphilus sp. gpac148 1451 AM176535 clade_434 N NFlexispira rappini 887 AY126479 clade_436 N N Helicobacter bilis 993ACDN01000023 clade_436 N N Helicobacter cinaedi 995 ABQT01000054clade_436 N N Helicobacter sp. None 998 U44756 clade_436 N NBrevundimonas subvibrioides 429 CP002102 clade_438 N N Hyphomonasneptunium 1008 NR_074092 clade_438 N N Phenylobacterium zucineum 1465AY628697 clade_438 N N Streptococcus downei 1793 AEKN01000002 clade_441N N Streptococcus sp. SHV515 1848 Y07601 clade_441 N N Acinetobacter sp.CIP 53.82 40 JQ638584 clade_443 N N Halomonas elongata 990 NR_074782clade_443 N N Halomonas johnsoniae 991 FR775979 clade_443 N NButyrivibrio fibrisolvens 456 U41172 clade_444 N N Roseburia sp. 11SE371640 FM954975 clade_444 N N Roseburia sp. 11SE38 1641 FM954976 clade_444N N Shuttleworthia satelles 1728 ACIP02000004 clade_444 N NShuttleworthia sp. MSX8B 1729 HQ616383 clade_444 N N Shuttleworthia sp.oral taxon G69 1730 GU432167 clade_444 N N Bdellovibrio sp. MPA 344AY294215 clade_445 N N Desulfobulbus sp. oral clone CH031 755 AY005036clade_445 N N Desulfovibrio desulfuricans 757 DQ092636 clade_445 N NDesulfovibrio fairfieldensis 758 U42221 clade_445 N N Desulfovibriopiger 759 AF192152 clade_445 N N Desulfovibrio sp. 3_1_syn3 760ADDR01000239 clade_445 N N Geobacter bemidjiensis 941 CP001124 clade_445N N Brachybacterium alimentarium 401 NR_026269 clade_446 N NBrachybacterium conglomeratum 402 AB537169 clade_446 N N Brachybacteriumtyrofermentans 403 NR_026272 clade_446 N N Dermabacter hominis 749FJ263375 clade_446 N N Aneurinibacillus thermoaerophilus 171 NR_029303clade_448 N N Brevibacillus agri 409 NR_040983 clade_448 N NBrevibacillus centrosporus 411 NR_043414 clade_448 N N Brevibacilluschoshinensis 412 NR_040980 clade_448 N N Brevibacillus invocatus 413NR_041836 clade_448 N N Brevibacillus parabrevis 415 NR_040981 clade_448N N Brevibacillus reuszeri 416 NR_040982 clade_448 N N Brevibacillus sp.phR 417 JN837488 clade_448 N N Brevibacillus thermoruber 418 NR_026514clade_448 N N Lactobacillus murinus 1100 NR_042231 clade_449 N NLactobacillus oeni 1102 NR_043095 clade_449 N N Lactobacillus ruminis1115 ACGS02000043 clade_449 N N Lactobacillus vini 1141 NR_042196clade_449 N N Gemella haemolysans 924 ACDZ02000012 clade_450 N N Gemellamorbillorum 925 NR_025904 clade_450 N N Gemella morbillorum 926ACRX01000010 clade_450 N N Gemella sanguinis 927 ACRY01000057 clade_450N N Gemella sp. oral clone ASCE02 929 AY923133 clade_450 N N Gemella sp.oral clone ASCF04 930 AY923139 clade_450 N N Gemella sp. oral cloneASCF12 931 AY923143 clade_450 N N Gemella sp. WAL 1945J 928 EU427463clade_450 N N Sporolactobacillus nakayamae 1753 NR_042247 clade_451 N NGluconacetobacter entanii 945 NR_028909 clade_452 N N Gluconacetobactereuropaeus 946 NR_026513 clade_452 N N Gluconacetobacter hansenii 947NR_026133 clade_452 N N Gluconacetobacter oboediens 949 NR_041295clade_452 N N Gluconacetobacter xylinus 950 NR_074338 clade_452 N NAuritibacter ignavus 193 FN554542 clade_453 N N Dermacoccus sp. Ellin185750 AEIQ01000090 clade_453 N N Janibacter limosus 1013 NR_026362clade_453 N N Janibacter melonis 1014 EF063716 clade_453 N N Acetobacteraceti 7 NR_026121 clade_454 N N Acetobacter fabarum 8 NR_042678clade_454 N N Acetobacter lovaniensis 9 NR_040832 clade_454 N NAcetobacter malorum 10 NR_025513 clade_454 N N Acetobacter orientalis 11NR_028625 clade_454 N N Acetobacter pasteurianus 12 NR_026107 clade_454N N Acetobacter pomorum 13 NR_042112 clade_454 N N Acetobacter syzygii14 NR_040868 clade_454 N N Acetobacter tropicalis 15 NR_036881 clade_454N N Gluconacetobacter azotocaptans 943 NR_028767 clade_454 N NGluconacetobacter diazotrophicus 944 NR_074292 clade_454 N NGluconacetobacter johannae 948 NR_024959 clade_454 N N Nocardiabrasiliensis 1351 AIHV01000038 clade_455 N N Nocardia cyriacigeorgica1352 HQ009486 clade_455 N N Nocardia puris 1354 NR_028994 clade_455 N NNocardia sp. 01_Je_025 1355 GU574059 clade_455 N N Rhodococcus equi 1623ADNW01000058 clade_455 N N Oceanobacillus caeni 1358 NR_041533 clade_456N N Oceanobacillus sp. Ndiop 1359 CAER01000083 clade_456 N NOrnithinibacillus bavariensis 1384 NR_044923 clade_456 N NOrnithinibacillus sp. 7_10AIA 1385 FN397526 clade_456 N N Virgibacillusproomii 2005 NR_025308 clade_456 N N Corynebacterium amycolatum 683ABZU01000033 clade_457 N OP Corynebacterium hansenii 702 AM946639clade_457 N N Corynebacterium xerosis 735 FN179330 clade_457 N OPStaphylococcaceae bacterium NML 92_0017 1756 AY841362 clade_458 N NStaphylococcus fleurettii 1766 NR_041326 clade_458 N N Staphylococcussciuri 1774 NR_025520 clade_458 N N Staphylococcus vitulinus 1779NR_024670 clade_458 N N Stenotrophomonas maltophilia 1782 AAVZ01000005clade_459 N N Stenotrophomonas sp. FG_6 1783 EF017810 clade_459 N NMycobacterium africanum 1270 AF480605 clade_46 N OP Mycobacteriumalsiensis 1271 AJ938169 clade_46 N OP Mycobacterium avium 1272 CP000479clade_46 N OP Mycobacterium colombiense 1274 AM062764 clade_46 N OPMycobacterium gordonae 1276 GU142930 clade_46 N OP Mycobacteriumintracellulare 1277 GQ153276 clade_46 N OP Mycobacterium kansasii 1278AF480601 clade_46 N OP Mycobacterium lacus 1279 NR_025175 clade_46 N OPMycobacterium leprae 1280 FM211192 clade_46 N OP Mycobacteriumlepromatosis 1281 EU203590 clade_46 N OP Mycobacterium mantenii 1283FJ042897 clade_46 N OP Mycobacterium marinum 1284 NC_010612 clade_46 NOP Mycobacterium microti 1285 NR_025234 clade_46 N OP Mycobacteriumparascrofulaceum 1287 ADNV01000350 clade_46 N OP Mycobacterium seoulense1290 DQ536403 clade_46 N OP Mycobacterium sp. 1761 1292 EU703150clade_46 N N Mycobacterium sp. 1791 1295 EU703148 clade_46 N NMycobacterium sp. 1797 1296 EU703149 clade_46 N N Mycobacterium sp.B10_07.09.0206 1298 HQ174245 clade_46 N N Mycobacterium sp. NLA0010007361305 HM627011 clade_46 N N Mycobacterium sp. W 1306 DQ437715 clade_46 NN Mycobacterium tuberculosis 1307 CP001658 clade_46 N Category-CMycobacterium ulcerans 1308 AB548725 clade_46 N OP Mycobacteriumvulneris 1309 EU834055 clade_46 N OP Xanthomonas campestris 2016EF101975 clade_461 N N Xanthomonas sp. kmd_489 2017 EU723184 clade_461 NN Dietzia natronolimnaea 769 GQ870426 clade_462 N N Dietzia sp. BBDP51770 DQ337512 clade_462 N N Dietzia sp. CA149 771 GQ870422 clade_462 N NDietzia timorensis 772 GQ870424 clade_462 N N Gordonia bronchialis 951NR_027594 clade_463 N N Gordonia polyisoprenivorans 952 DQ385609clade_463 N N Gordonia sp. KTR9 953 DQ068383 clade_463 N N Gordoniasputi 954 FJ536304 clade_463 N N Gordonia terrae 955 GQ848239 clade_463N N Leptotrichia goodfellowii 1167 ADAD01000110 clade_465 N NLeptotrichia sp. oral clone IK040 1174 AY349387 clade_465 N NLeptotrichia sp. oral clone P2PB_51 P1 1175 AY207053 clade_465 N NBacteroidales genomosp. P7 oral clone MB3_P19 264 DQ003623 clade_466 N NButyricimonas virosa 454 AB443949 clade_466 N N Odoribacter laneus 1363AB490805 clade_466 N N Odoribacter splanchnicus 1364 CP002544 clade_466N N Capnocytophaga gingivalis 478 ACLQ01000011 clade_467 N NCapnocytophaga granulosa 479 X97248 clade_467 N N Capnocytophaga sp.oral clone AH015 483 AY005074 clade_467 N N Capnocytophaga sp. oralstrain S3 487 AY005073 clade_467 N N Capnocytophaga sp. oral taxon 338488 AEXX01000050 clade_467 N N Capnocytophaga canimorsus 476 CP002113clade_468 N N Capnocytophaga sp. oral clone ID062 485 AY349368 clade_468N N Lactobacillus catenaformis 1075 M23729 clade_469 N N Lactobacillusvitulinus 1142 NR_041305 clade_469 N N Cetobacterium somerae 501AJ438155 clade_470 N N Fusobacterium gonidiaformans 896 ACET01000043clade_470 N N Fusobacterium mortiferum 897 ACDB02000034 clade_470 N NFusobacterium necrogenes 899 X55408 clade_470 N N Fusobacteriumnecrophorum 900 AM905356 clade_470 N N Fusobacterium sp. 12_1B 905AGWJ01000070 clade_470 N N Fusobacterium sp. 3_1_5R 911 ACDD01000078clade_470 N N Fusobacterium sp. D12 918 ACDG02000036 clade_470 N NFusobacterium ulcerans 921 ACDH01000090 clade_470 N N Fusobacteriumvarium 922 ACIE01000009 clade_470 N N Mycoplasma arthritidis 1312NC_011025 clade_473 N N Mycoplasma faucium 1314 NR_024983 clade_473 N NMycoplasma hominis 1318 AF443616 clade_473 N N Mycoplasma orale 1319AY796060 clade_473 N N Mycoplasma salivarium 1324 M24661 clade_473 N NMitsuokella jalaludinii 1247 NR_028840 clade_474 N N Mitsuokellamultacida 1248 ABWK02000005 clade_474 N N Mitsuokella sp. oral taxon 5211249 GU413658 clade_474 N N Mitsuokella sp. oral taxon G68 1250 GU432166clade_474 N N Selenomonas genomosp. C1 1695 AY278627 clade_474 N NSelenomonas genomosp. P8 oral clone MB5_P06 1700 DQ003628 clade_474 N NSelenomonas ruminantium 1703 NR_075026 clade_474 N N Veillonellaceaebacterium oral taxon 131 1994 GU402916 clade_474 N N Alloscardoviaomnicolens 139 NR_042583 clade_475 N N Alloscardovia sp. OB7196 140AB425070 clade_475 N N Bifidobacterium urinalis 366 AJ278695 clade_475 NN Prevotella loescheii 1503 JN867231 clade_48 N N Prevotella sp. oralclone ASCG12 1530 DQ272511 clade_48 N N Prevotella sp. oral clone GU0271540 AY349398 clade_48 N N Prevotella sp. oral taxon 472 1553ACZS01000106 clade_48 N N Selenomonas dianae 1693 GQ422719 clade_480 N NSelenomonas flueggei 1694 AF287803 clade_480 N N Selenomonas genomosp.C2 1696 AY278628 clade_480 N N Selenomonas genomosp. P6 oral cloneMB3_C41 1698 DQ003636 clade_480 N N Selenomonas genomosp. P7 oral cloneMB5_C08 1699 DQ003627 clade_480 N N Selenomonas infelix 1701 AF287802clade_480 N N Selenomonas noxia 1702 GU470909 clade_480 N N Selenomonassp. oral clone FT050 1705 AY349403 clade_480 N N Selenomonas sp. oralclone GI064 1706 AY349404 clade_480 N N Selenomonas sp. oral clone GT0101707 AY349405 clade_480 N N Selenomonas sp. oral clone HU051 1708AY349406 clade_480 N N Selenomonas sp. oral clone IK004 1709 AY349407clade_480 N N Selenomonas sp. oral clone JI021 1711 AY349409 clade_480 NN Selenomonas sp. oral clone JS031 1712 AY349410 clade_480 N NSelenomonas sp. oral clone OH4A 1713 AY947498 clade_480 N N Selenomonassp. oral clone P2PA_80 P4 1714 AY207052 clade_480 N N Selenomonas sp.oral taxon 149 1716 AEEJ01000007 clade_480 N N Veillonellaceae bacteriumoral taxon 155 1995 GU470897 clade_480 N N Agrococcus jenensis 117NR_026275 clade_484 N N Microbacterium gubbeenense 1232 NR_025098clade_484 N N Pseudoclavibacter sp. Timone 1590 FJ375951 clade_484 N NTropheryma whipplei 1961 BX251412 clade_484 N N Zimmermannella bifida2031 AB012592 clade_484 N N Legionella hackeliae 1151 M36028 clade_486 NOP Legionella longbeachae 1152 M36029 clade_486 N OP Legionella sp.D3923 1154 JN380999 clade_486 N OP Legionella sp. D4088 1155 JN381012clade_486 N OP Legionella sp. H63 1156 JF831047 clade_486 N OPLegionella sp. NML 93L054 1157 GU062706 clade_486 N OP Legionellasteelei 1158 HQ398202 clade_486 N OP Tatlockia micdadei 1915 M36032clade_486 N N Helicobacter pullorum 996 ABQU01000097 clade_489 N NAcetobacteraceae bacterium AT 5844 16 AGEZ01000040 clade_490 N NRoseomonas cervicalis 1643 ADVL01000363 clade_490 N N Roseomonas mucosa1644 NR_028857 clade_490 N N Roseomonas sp. NML94_0193 1645 AF533357clade_490 N N Roseomonas sp. NML97_0121 1646 AF533359 clade_490 N NRoseomonas sp. NML98_0009 1647 AF533358 clade_490 N N Roseomonas sp.NML98_0157 1648 AF533360 clade_490 N N Rickettsia akari 1627 CP000847clade_492 N OP Rickettsia conorii 1628 AE008647 clade_492 N OPRickettsia prowazekii 1629 M21789 clade_492 N Category-B Rickettsiarickettsii 1630 NC_010263 clade_492 N OP Rickettsia slovaca 1631 L36224clade_492 N OP Rickettsia typhi 1632 AE017197 clade_492 N OPAnaeroglobus geminatus 160 AGCJ01000054 clade_493 N N Megasphaeragenomosp. C1 1201 AY278622 clade_493 N N Megasphaera micronuciformis1203 AECS01000020 clade_493 N N Clostridiales genomosp. BVAB3 540CP001850 clade_495 N N Tsukamurella paurometabola 1963 X80628 clade_496N N Tsukamurella tyrosinosolvens 1964 AB478958 clade_496 N N Abiotrophiapara_adiacens 2 AB022027 clade_497 N N Carnobacterium divergens 492NR_044706 clade_497 N N Carnobacterium maltaromaticum 493 NC_019425clade_497 N N Enterococcus avium 800 AF133535 clade_497 N N Enterococcuscaccae 801 AY943820 clade_497 N N Enterococcus casseliflavus 802AEWT01000047 clade_497 N N Enterococcus durans 803 AJ276354 clade_497 NN Enterococcus faecalis 804 AE016830 clade_497 N N Enterococcus faecium805 AM157434 clade_497 N N Enterococcus gallinarum 806 AB269767clade_497 N N Enterococcus gilvus 807 AY033814 clade_497 N NEnterococcus hawaiiensis 808 AY321377 clade_497 N N Enterococcus hirae809 AF061011 clade_497 N N Enterococcus italicus 810 AEPV01000109clade_497 N N Enterococcus mundtii 811 NR_024906 clade_497 N NEnterococcus raffinosus 812 FN600541 clade_497 N N Enterococcus sp.BV2CASA2 813 JN809766 clade_497 N N Enterococcus sp. CCRI_16620 814GU457263 clade_497 N N Enterococcus sp. F95 815 FJ463817 clade_497 N NEnterococcus sp. RfL6 816 AJ133478 clade_497 N N Enterococcusthailandicus 817 AY321376 clade_497 N N Fusobacterium canifelinum 893AY162222 clade_497 N N Fusobacterium genomosp. C1 894 AY278616 clade_497N N Fusobacterium genomosp. C2 895 AY278617 clade_497 N N Fusobacteriumperiodonticum 902 ACJY01000002 clade_497 N N Fusobacterium sp. 1_1_41FAA906 ADGG01000053 clade_497 N N Fusobacterium sp. 11_3_2 904 ACUO01000052clade_497 N N Fusobacterium sp. 2_1_31 907 ACDC02000018 clade_497 N NFusobacterium sp. 3_1_27 908 ADGF01000045 clade_497 N N Fusobacteriumsp. 3_1_33 909 ACQE01000178 clade_497 N N Fusobacterium sp. 3_1_36A2 910ACPU01000044 clade_497 N N Fusobacterium sp. AC18 912 HQ616357 clade_497N N Fusobacterium sp. ACB2 913 HQ616358 clade_497 N N Fusobacterium sp.AS2 914 HQ616361 clade_497 N N Fusobacterium sp. CM1 915 HQ616371clade_497 N N Fusobacterium sp. CM21 916 HQ616375 clade_497 N NFusobacterium sp. CM22 917 HQ616376 clade_497 N N Fusobacterium sp. oralclone ASCF06 919 AY923141 clade_497 N N Fusobacterium sp. oral cloneASCF11 920 AY953256 clade_497 N N Granulicatella adiacens 959ACKZ01000002 clade_497 N N Granulicatella elegans 960 AB252689 clade_497N N Granulicatella paradiacens 961 AY879298 clade_497 N N Granulicatellasp. oral clone ASC02 963 AY923126 clade_497 N N Granulicatella sp. oralclone ASCA05 964 DQ341469 clade_497 N N Granulicatella sp. oral cloneASCB09 965 AY953251 clade_497 N N Granulicatella sp. oral clone ASCG05966 AY923146 clade_497 N N Tetragenococcus halophilus 1918 NR_075020clade_497 N N Tetragenococcus koreensis 1919 NR_043113 clade_497 N NVagococcus fluvialis 1973 NR_026489 clade_497 N N Chryseobacteriumanthropi 514 AM982793 clade_498 N N Chryseobacterium gleum 515ACKQ02000003 clade_498 N N Chryseobacterium hominis 516 NR_042517clade_498 N N Treponema refringens 1936 AF426101 clade_499 N OPTreponema sp. oral clone JU031 1941 AY349416 clade_499 N N Treponema sp.oral taxon 239 1948 GU408738 clade_499 N N Treponema sp. oral taxon 2711955 GU408871 clade_499 N N Alistipes finegoldii 129 NR_043064 clade_500N N Alistipes onderdonkii 131 NR_043318 clade_500 N N Alistipesputredinis 132 ABFK02000017 clade_500 N N Alistipes shahii 133 FP929032clade_500 N N Alistipes sp. HGB5 134 AENZ01000082 clade_500 N NAlistipes sp. JC50 135 JF824804 clade_500 N N Alistipes sp. RMA 9912 136GQ140629 clade_500 N N Mycoplasma agalactiae 1310 AF010477 clade_501 N NMycoplasma bovoculi 1313 NR_025987 clade_501 N N Mycoplasma fermentans1315 CP002458 clade_501 N N Mycoplasma flocculare 1316 X62699 clade_501N N Mycoplasma ovipneumoniae 1320 NR_025989 clade_501 N N Arcobacterbutzleri 176 AEPT01000071 clade_502 N N Arcobacter cryaerophilus 177NR_025905 clade_502 N N Campylobacter curvus 461 NC_009715 clade_502 NOP Campylobacter rectus 467 ACFU01000050 clade_502 N OP Campylobactershowae 468 ACVQ01000030 clade_502 N OP Campylobacter sp. FOBRC14 469HQ616379 clade_502 N OP Campylobacter sp. FOBRC15 470 HQ616380 clade_502N OP Campylobacter sp. oral clone BB120 471 AY005038 clade_502 N OPCampylobacter sputorum 472 NR_044839 clade_502 N OP Bacteroidesureolyticus 330 GQ167666 clade_504 N N Campylobacter gracilis 463ACYG01000026 clade_504 N OP Campylobacter hominis 464 NC_009714clade_504 N OP Dialister invisus 762 ACIM02000001 clade_506 N NDialister micraerophilus 763 AFBB01000028 clade_506 N N Dialistermicroaerophilus 764 AENT01000008 clade_506 N N Dialisterpropionicifaciens 766 NR_043231 clade_506 N N Dialister succinatiphilus768 AB370249 clade_506 N N Megasphaera elsdenii 1200 AY038996 clade_506N N Megasphaera genomosp. type_1 1202 ADGP01000010 clade_506 N NMegasphaera sp. BLPYG_07 1204 HM990964 clade_506 N N Megasphaera sp.UPII 199_6 1205 AFIJ01000040 clade_506 N N Chromobacterium violaceum 513NC_005085 clade_507 N N Laribacter hongkongensis 1148 CP001154 clade_507N N Methylophilus sp. ECd5 1229 AY436794 clade_507 N N Finegoldia magna883 ACHM02000001 clade_509 N N Parvimonas micra 1431 AB729072 clade_509N N Parvimonas sp. oral taxon 110 1432 AFII01000002 clade_509 N NPeptostreptococcus micros 1456 AM176538 clade_509 N N Peptostreptococcussp. oral clone FJ023 1460 AY349390 clade_509 N N Peptostreptococcus sp.P4P_31 P3 1458 AY207059 clade_509 N N Helicobacter pylori 997 CP000012clade_510 N OP Anaplasma marginale 165 ABOR01000019 clade_511 N NAnaplasma phagocytophilum 166 NC_007797 clade_511 N N Ehrlichiachaffeensis 783 AAIF01000035 clade_511 N OP Neorickettsia risticii 1349CP001431 clade_511 N N Neorickettsia sennetsu 1350 NC_007798 clade_511 NN Pseudoramibacter alactolyticus 1606 AB036759 clade_512 N N Veillonellamontpellierensis 1977 AF473836 clade_513 N N Veillonella sp. oral cloneASCA08 1988 AY923118 clade_513 N N Veillonella sp. oral clone ASCB031989 AY923122 clade_513 N N Inquilinus limosus 1012 NR_029046 clade_514N N Sphingomonas sp. oral clone FZ016 1746 AY349412 clade_514 N NAnaerococcus lactolyticus 145 ABYO01000217 clade_515 N N Anaerococcusprevotii 147 CP001708 clade_515 N N Anaerococcus sp. gpac104 152AM176528 clade_515 N N Anaerococcus sp. gpac126 153 AM176530 clade_515 NN Anaerococcus sp. gpac155 154 AM176536 clade_515 N N Anaerococcus sp.gpac199 155 AM176539 clade_515 N N Anaerococcus tetradius 157ACGC01000107 clade_515 N N Bacteroides coagulans 271 AB547639 clade_515N N Clostridiales bacterium 9403326 534 HM587324 clade_515 N NClostridiales bacterium ph2 539 JN837487 clade_515 N NPeptostreptococcus sp. 9succ1 1457 X90471 clade_515 N NPeptostreptococcus sp. oral clone AP24 1459 AB175072 clade_515 N NTissierella praeacuta 1924 NR_044860 clade_515 N N Helicobactercanadensis 994 ABQS01000108 clade_518 N N Peptostreptococcus anaerobius1455 AY326462 clade_520 N N Peptostreptococcus stomatis 1461ADGQ01000048 clade_520 N N Bilophila wadsworthia 367 ADCP01000166clade_521 N N Desulfovibrio vulgaris 761 NR_074897 clade_521 N NActinomyces nasicola 64 AJ508455 clade_523 N N Cellulosimicrobium funkei500 AY501364 clade_523 N N Lactococcus raffinolactis 1146 NR_044359clade_524 N N Bacteroidales genomosp. P1 258 AY341819 clade_529 N NBacteroidales genomosp. P2 oral clone MB1_G13 259 DQ003613 clade_529 N NBacteroidales genomosp. P3 oral clone MB1_G34 260 DQ003615 clade_529 N NBacteroidales genomosp. P4 oral clone MB2_G17 261 DQ003617 clade_529 N NBacteroidales genomosp. P5 oral clone MB2_P04 262 DQ003619 clade_529 N NBacteroidales genomosp. P6 oral clone MB3_C19 263 DQ003634 clade_529 N NBacteroidales genomosp. P8 oral clone MB4_G15 265 DQ003626 clade_529 N NBacteroidetes bacterium oral taxon D27 333 HM099638 clade_530 N NBacteroidetes bacterium oral taxon F31 334 HM099643 clade_530 N NBacteroidetes bacterium oral taxon F44 335 HM099649 clade_530 N NFlavobacterium sp. NF2_1 885 FJ195988 clade_530 N N Myroidesodoratimimus 1326 NR_042354 clade_530 N N Myroides sp. MY15 1327GU253339 clade_530 N N Chlamydiales bacterium NS16 507 JN606076clade_531 N N Chlamydophila pecorum 508 D88317 clade_531 N OPParachlamydia sp. UWE25 1423 BX908798 clade_531 N N Fusobacterium russii903 NR_044687 clade_532 N N Streptobacillus moniliformis 1784 NR_027615clade_532 N N Eubacteriaceae bacterium P4P_50 P4 833 AY207060 clade_533N N Abiotrophia defectiva 1 ACIN02000016 clade_534 N N Abiotrophia sp.oral clone P4PA_155 P1 3 AY207063 clade_534 N N Catonella genomosp. P1oral clone MB5_P12 496 DQ003629 clade_534 N N Catonella morbi 497ACIL02000016 clade_534 N N Catonella sp. oral clone FL037 498 AY349369clade_534 N N Eremococcus coleocola 818 AENN01000008 clade_534 N NFacklamia hominis 879 Y10772 clade_534 N N Granulicatella sp. M658_99_3962 AJ271861 clade_534 N N Campylobacter coli 459 AAFL01000004 clade_535N OP Campylobacter concisus 460 CP000792 clade_535 N OP Campylobacterfetus 462 ACLG01001177 clade_535 N OP Campylobacter jejuni 465 AL139074clade_535 N Category-B Campylobacter upsaliensis 473 AEPU01000040clade_535 N OP Atopobium minutum 183 HM007583 clade_539 N N Atopobiumparvulum 184 CP001721 clade_539 N N Atopobium rimae 185 ACFE01000007clade_539 N N Atopobium sp. BS2 186 HQ616367 clade_539 N N Atopobium sp.F0209 187 EU592966 clade_539 N N Atopobium sp. ICM42b10 188 HQ616393clade_539 N N Atopobium sp. ICM57 189 HQ616400 clade_539 N N Atopobiumvaginae 190 AEDQ01000024 clade_539 N N Coriobacteriaceae bacteriumBV3Ac1 677 JN809768 clade_539 N N Actinomyces naeslundii 63 X81062clade_54 N N Actinomyces oricola 67 NR_025559 clade_54 N N Actinomycesoris 69 BABV01000070 clade_54 N N Actinomyces sp. 7400942 70 EU484334clade_54 N N Actinomyces sp. ChDC B197 72 AF543275 clade_54 N NActinomyces sp. GEJ15 73 GU561313 clade_54 N N Actinomyces sp.M2231_94_1 79 AJ234063 clade_54 N N Actinomyces sp. oral clone GU067 83AY349362 clade_54 N N Actinomyces sp. oral clone IO077 85 AY349364clade_54 N N Actinomyces sp. oral clone IP073 86 AY349365 clade_54 N NActinomyces sp. oral clone JA063 88 AY349367 clade_54 N N Actinomycessp. oral taxon 170 89 AFBL01000010 clade_54 N N Actinomyces sp. oraltaxon 171 90 AECW01000034 clade_54 N N Actinomyces urogenitalis 95ACFH01000038 clade_54 N N Actinomyces viscosus 96 ACRE01000096 clade_54N N Orientia tsutsugamushi 1383 AP008981 clade_541 N OP Megamonasfuniformis 1198 AB300988 clade_542 N N Megamonas hypermegale 1199AJ420107 clade_542 N N Aeromicrobium marinum 102 NR_025681 clade_544 N NAeromicrobium sp. JC14 103 JF824798 clade_544 N N Luteococcus sanguinis1190 NR_025507 clade_544 N N Propionibacteriaceae bacterium NML 02_02651568 EF599122 clade_544 N N Rhodococcus corynebacterioides 1622 X80615clade_546 N N Rhodococcus erythropolis 1624 ACNO01000030 clade_546 N NRhodococcus fascians 1625 NR_037021 clade_546 N N Segniliparus rotundus1690 CP001958 clade_546 N N Segniliparus rugosus 1691 ACZI01000025clade_546 N N Exiguobacterium acetylicum 878 FJ970034 clade_547 N NMacrococcus caseolyticus 1194 NR_074941 clade_547 N N Streptomyces sp. 1AIP_2009 1890 FJ176782 clade_548 N N Streptomyces sp. SD 524 1892EU544234 clade_548 N N Streptomyces sp. SD 528 1893 EU544233 clade_548 NN Streptomyces thermoviolaceus 1895 NR_027616 clade_548 N N Borreliaafzelii 388 ABCU01000001 clade_549 N OP Borrelia crocidurae 390 DQ057990clade_549 N OP Borrelia duttonii 391 NC_011229 clade_549 N OP Borreliahermsii 393 AY597657 clade_549 N OP Borrelia hispanica 394 DQ057988clade_549 N OP Borrelia persica 395 HM161645 clade_549 N OP Borreliarecurrentis 396 AF107367 clade_549 N OP Borrelia spielmanii 398ABKB01000002 clade_549 N OP Borrelia turicatae 399 NC_008710 clade_549 NOP Borrelia valaisiana 400 ABCY01000002 clade_549 N OP Providenciaalcalifaciens 1586 ABXW01000071 clade_55 N N Providencia rettgeri 1587AM040492 clade_55 N N Providencia rustigianii 1588 AM040489 clade_55 N NProvidencia stuartii 1589 AF008581 clade_55 N N Treponema pallidum 1932CP001752 clade_550 N OP Treponema phagedenis 1934 AEFH01000172 clade_550N N Treponema sp. clone DDKL_4 1939 Y08894 clade_550 N N Acholeplasmalaidlawii 17 NR_074448 clade_551 N N Mycoplasma putrefaciens 1323 U26055clade_551 N N Mycoplasmataceae genomosp. P1 oral clone MB1_G23 1325DQ003614 clade_551 N N Spiroplasma insolitum 1750 NR_025705 clade_551 NN Collinsella intestinalis 660 ABXH02000037 clade_553 N N Collinsellastercoris 661 ABXJ01000150 clade_553 N N Collinsella tanakaei 662AB490807 clade_553 N N Caminicella sporogenes 458 NR_025485 clade_554 NN Acidaminococcus fermentans 21 CP001859 clade_556 N N Acidaminococcusintestini 22 CP003058 clade_556 N N Acidaminococcus sp. D21 23ACGB01000071 clade_556 N N Phascolarctobacterium faecium 1462 NR_026111clade_556 N N Phascolarctobacterium sp. YIT 12068 1463 AB490812clade_556 N N Phascolarctobacterium succinatutens 1464 AB490811clade_556 N N Acidithiobacillus ferrivorans 25 NR_074660 clade_557 N NXanthomonadaceae bacterium NML 03_0222 2015 EU313791 clade_557 N NCatabacter hongkongensis 494 AB671763 clade_558 N N Christensenellaminuta 512 AB490809 clade_558 N N Clostridiales bacterium oral cloneP4PA_66 P1 536 AY207065 clade_558 N N Clostridiales bacterium oral taxon093 537 GQ422712 clade_558 N N Heliobacterium modesticaldum 1000NR_074517 clade_560 N N Alistipes indistinctus 130 AB490804 clade_561 NN Bacteroidales bacterium ph 8 257 JN837494 clade_561 N N CandidatesSulcia muelleri 475 CP002163 clade_561 N N Cytophaga xylanolytica 742FR733683 clade_561 N N Flavobacteriaceae genomosp. C1 884 AY278614clade_561 N N Gramella forsetii 958 NR_074707 clade_561 N NSphingobacterium faecium 1740 NR_025537 clade_562 N N Sphingobacteriummizutaii 1741 JF708889 clade_562 N N Sphingobacterium multivorum 1742NR_040953 clade_562 N N Sphingobacterium spiritivorum 1743 ACHA02000013clade_562 N N Jonquetella anthropi 1017 ACOO02000004 clade_563 N NPyramidobacter piscolens 1614 AY207056 clade_563 N N Synergistesgenomosp. C1 1904 AY278615 clade_563 N N Synergistes sp. RMA 14551 1905DQ412722 clade_563 N N Synergistetes bacterium ADV897 1906 GQ258968clade_563 N N Candidates Arthromitus sp. 474 NR_074460 clade_564 N NSFB_mouse_Yit Gracilibacter thermotolerans 957 NR_043559 clade_564 N NBrachyspira aalborgi 404 FM178386 clade_565 N N Brachyspira sp. HIS3 406FM178387 clade_565 N N Brachyspira sp. HIS4 407 FM178388 clade_565 N NBrachyspira sp. HIS5 408 FM178389 clade_565 N N Adlercreutziaequolifaciens 97 AB306661 clade_566 N N Coriobacteriaceae bacteriumJC110 678 CAEM01000062 clade_566 N N Coriobacteriaceae bacterium phI 679JN837493 clade_566 N N Cryptobacterium curtum 740 GQ422741 clade_566 N NEggerthella sinensis 779 AY321958 clade_566 N N Eggerthella sp.1_3_56FAA 780 ACWN01000099 clade_566 N N Eggerthella sp. HGA1 781AEXR01000021 clade_566 N N Eggerthella sp. YY7918 782 AP012211 clade_566N N Gordonibacter pamelaeae 680 AM886059 clade_566 N N Gordonibacterpamelaeae 956 FP929047 clade_566 N N Slackia equolifaciens 1732 EU377663clade_566 N N Slackia exigua 1733 ACUX01000029 clade_566 N N Slackiafaecicanis 1734 NR_042220 clade_566 N N Slackia heliotrinireducens 1735NR_074439 clade_566 N N Slackia isoflavoniconvertens 1736 AB566418clade_566 N N Slackia piriformis 1737 AB490806 clade_566 N N Slackia sp.NATTS 1738 AB505075 clade_566 N N Chlamydiales bacterium NS13 506JN606075 clade_567 N N Victivallaceae bacterium NML 080035 2003 FJ394915clade_567 N N Victivallis vadensis 2004 ABDE02000010 clade_567 N NStreptomyces griseus 1889 NR_074787 clade_573 N N Streptomyces sp. SD511 1891 EU544231 clade_573 N N Streptomyces sp. SD 534 1894 EU544232clade_573 N N Cloacibacillus evryensis 530 GQ258966 clade_575 N NDeferribacteres sp. oral clone JV001 743 AY349370 clade_575 N NDeferribacteres sp. oral clone JV023 745 AY349372 clade_575 N NSynergistetes bacterium LBVCM1157 1907 GQ258969 clade_575 N NSynergistetes bacterium oral taxon 362 1909 GU410752 clade_575 N NSynergistetes bacterium oral taxon D48 1910 GU430992 clade_575 N NPeptococcus sp. oral clone JM048 1439 AY349389 clade_576 N NHelicobacter winghamensis 999 ACDO01000013 clade_577 N N Wolinellasuccinogenes 2014 BX571657 clade_577 N N Olsenella genomosp. C1 1368AY278623 clade_578 N N Olsenella profusa 1369 FN178466 clade_578 N NOlsenella sp. F0004 1370 EU592964 clade_578 N N Olsenella sp. oral taxon809 1371 ACVE01000002 clade_578 N N Olsenella uli 1372 CP002106clade_578 N N Nocardiopsis dassonvillei 1356 CP002041 clade_579 N NPeptococcus niger 1438 NR_029221 clade_580 N N Peptococcus sp. oraltaxon 167 1440 GQ422727 clade_580 N N Akkermansia muciniphila 118CP001071 clade_583 N N Opitutus terrae 1373 NR_074978 clade_583 N NClostridiales bacterium oral taxon F32 538 HM099644 clade_584 N NLeptospira borgpetersenii 1161 NC_008508 clade_585 N OP Leptospirabroomii 1162 NR_043200 clade_585 N OP Leptospira interrogans 1163NC_005823 clade_585 N OP Methanobrevibacter gottschalkii 1213 NR_044789clade_587 N N Methanobrevibacter millerae 1214 NR_042785 clade_587 N NMethanobrevibacter oralis 1216 HE654003 clade_587 N N Methanobrevibacterthaueri 1219 NR_044787 clade_587 N N Methanobrevibacter smithii 1218ABYV02000002 clade_588 N N Deinococcus radiodurans 746 AE000513clade_589 N N Deinococcus sp. R_43890 747 FR682752 clade_589 N N Thermusaquaticus 1923 NR_025900 clade_589 N N Actinomyces sp. c109 81 AB167239clade_590 N N Syntrophomonadaceae genomosp. P1 1912 AY341821 clade_590 NN Anaerobaculum hydrogeniformans 141 ACJX02000009 clade_591 N NMicrocystis aeruginosa 1246 NC_010296 clade_592 N N Prochlorococcusmarinus 1567 CP000551 clade_592 N N Methanobrevibacter acididurans 1208NR_028779 clade_593 N N Methanobrevibacter arboriphilus 1209 NR_042783clade_593 N N Methanobrevibacter curvatus 1210 NR_044796 clade_593 N NMethanobrevibacter cuticularis 1211 NR_044776 clade_593 N NMethanobrevibacter filiformis 1212 NR_044801 clade_593 N NMethanobrevibacter woesei 1220 NR_044788 clade_593 N N Roseiflexuscastenholzii 1642 CP000804 clade_594 N N Methanobrevibacter olleyae 1215NR_043024 clade_595 N N Methanobrevibacter ruminantium 1217 NR_042784clade_595 N N Methanobrevibacter wolinii 1221 NR_044790 clade_595 N NMethanosphaera stadtmanae 1222 AY196684 clade_595 N N Chloroflexigenomosp. P1 511 AY331414 clade_596 N N Halorubrum lipolyticum 992AB477978 clade_597 N N Methanobacterium formicicum 1207 NR_025028clade_597 N N Acidilobus saccharovorans 24 AY350586 clade_598 N NHyperthermus butylicus 1006 CP000493 clade_598 N N Ignicoccus islandicus1011 X99562 clade_598 N N Metallosphaera sedula 1206 D26491 clade_598 NN Thermofilum pendens 1922 X14835 clade_598 N N Prevotellamelaninogenica 1506 CP002122 clade_6 N N Prevotella sp. ICM1 1520HQ616385 clade_6 N N Prevotella sp. oral clone FU048 1535 AY349393clade_6 N N Prevotella sp. oral clone GI030 1537 AY349395 clade_6 N NPrevotella sp. SEQ116 1526 JN867246 clade_6 N N Streptococcus anginosus1787 AECT01000011 clade_60 N N Streptococcus milleri 1812 X81023clade_60 N N Streptococcus sp. 16362 1829 JN590019 clade_60 N NStreptococcus sp. 69130 1832 X78825 clade_60 N N Streptococcus sp. AC151833 HQ616356 clade_60 N N Streptococcus sp. CM7 1839 HQ616373 clade_60N N Streptococcus sp. OBRC6 1847 HQ616352 clade_60 N N Burkholderiaambifaria 442 AAUZ01000009 clade_61 N OP Burkholderia cenocepacia 443AAHI01000060 clade_61 N OP Burkholderia cepacia 444 NR_041719 clade_61 NOP Burkholderia mallei 445 CP000547 clade_61 N Category-B Burkholderiamultivorans 446 NC_010086 clade_61 N OP Burkholderia oklahomensis 447DQ108388 clade_61 N OP Burkholderia pseudomallei 448 CP001408 clade_61 NCategory-B Burkholderia rhizoxinica 449 HQ005410 clade_61 N OPBurkholderia sp. 383 450 CP000151 clade_61 N OP Burkholderia xenovorans451 U86373 clade_61 N OP Prevotella buccae 1488 ACRB01000001 clade_62 NN Prevotella genomosp. P8 oral clone MB3_P13 1498 DQ003622 clade_62 N NPrevotella sp. oral clone FW035 1536 AY349394 clade_62 N N Prevotellabivia 1486 ADFO01000096 clade_63 N N Prevotella disiens 1494AEDO01000026 clade_64 N N Bacteroides faecis 276 GQ496624 clade_65 N NBacteroides fragilis 279 AP006841 clade_65 N N Bacteroides nordii 285NR_043017 clade_65 N N Bacteroides salyersiae 292 EU136690 clade_65 N NBacteroides sp. 1_1_14 293 ACRP01000155 clade_65 N N Bacteroides sp.1_1_6 295 ACIC01000215 clade_65 N N Bacteroides sp. 2_1_56FAA 298ACWI01000065 clade_65 N N Bacteroides sp. AR29 316 AF139525 clade_65 N NBacteroides sp. B2 317 EU722733 clade_65 N N Bacteroidesthetaiotaomicron 328 NR_074277 clade_65 N N Actinobacillus minor 45ACFT01000025 clade_69 N N Haemophilias parasuis 978 GU226366 clade_69 NN Vibrio cholerae 1996 AAUR01000095 clade_71 N Category-B Vibriofluvialis 1997 X76335 clade_71 N Category-B Vibrio furnissii 1998CP002377 clade_71 N Category-B Vibrio mimicus 1999 ADAF01000001 clade_71N Category-B Vibrio parahaemolyticus 2000 AAWQ01000116 clade_71 NCategory-B Vibrio sp. RC341 2001 ACZT01000024 clade_71 N Category-BVibrio vulnificus 2002 AE016796 clade_71 N Category-B Lactobacillusacidophilus 1067 CP000033 clade_72 N N Lactobacillus amylolyticus 1069ADNY01000006 clade_72 N N Lactobacillus amylovorus 1070 CP002338clade_72 N N Lactobacillus crispatus 1078 ACOG01000151 clade_72 N NLactobacillus delbrueckii 1080 CP002341 clade_72 N N Lactobacillushelveticus 1088 ACLM01000202 clade_72 N N Lactobacillus kalixensis 1094NR_029083 clade_72 N N Lactobacillus kefiranofaciens 1095 NR_042440clade_72 N N Lactobacillus leichmannii 1098 JX986966 clade_72 N NLactobacillus sp. 66c 1120 FR681900 clade_72 N N Lactobacillus sp. KLDS1.0701 1122 EU600905 clade_72 N N Lactobacillus sp. KLDS 1.0712 1130EU600916 clade_72 N N Lactobacillus sp. oral clone HT070 1136 AY349383clade_72 N N Lactobacillus ultunensis 1139 ACGU01000081 clade_72 N NPrevotella intermedia 1502 AF414829 clade_81 N N Prevotella nigrescens1511 AFPX01000069 clade_81 N N Prevotella pallens 1515 AFPY01000135clade_81 N N Prevotella sp. oral taxon 310 1551 GQ422737 clade_81 N NPrevotella genomosp. C1 1495 AY278624 clade_82 N N Prevotella sp. CM381519 HQ610181 clade_82 N N Prevotella sp. oral taxon 317 1552ACQH01000158 clade_82 N N Prevotella sp. SG12 1527 GU561343 clade_82 N NPrevotella denticola 1493 CP002589 clade_83 N N Prevotella genomosp. P7oral clone MB2_P31 1497 DQ003620 clade_83 N N Prevotella histicola 1501JN867315 clade_83 N N Prevotella multiformis 1508 AEWX01000054 clade_83N N Prevotella sp. JCM 6330 1522 AB547699 clade_83 N N Prevotella sp.oral clone GI059 1539 AY349397 clade_83 N N Prevotella sp. oral taxon782 1555 GQ422745 clade_83 N N Prevotella sp. oral taxon G71 1559GU432180 clade_83 N N Prevotella sp. SEQ065 1524 JN867234 clade_83 N NPrevotella veroralis 1565 ACVA01000027 clade_83 N N Bacteroidesacidifaciens 266 NR_028607 clade_85 N N Bacteroides cellulosilyticus 269ACCH01000108 clade_85 N N Bacteroides clarus 270 AFBM01000011 clade_85 NN Bacteroides eggerthii 275 ACWG01000065 clade_85 N N Bacteroidesoleiciplenus 286 AB547644 clade_85 N N Bacteroides pyogenes 290NR_041280 clade_85 N N Bacteroides sp. 315_5 300 FJ848547 clade_85 N NBacteroides sp. 31SF15 301 AJ583248 clade_85 N N Bacteroides sp. 31SF18302 AJ583249 clade_85 N N Bacteroides sp. 35AE31 303 AJ583244 clade_85 NN Bacteroides sp. 35AE37 304 AJ583245 clade_85 N N Bacteroides sp.35BE34 305 AJ583246 clade_85 N N Bacteroides sp. 35BE35 306 AJ583247clade_85 N N Bacteroides sp. WH2 324 AY895180 clade_85 N N Bacteroidessp. XB12B 325 AM230648 clade_85 N N Bacteroides stercoris 327ABFZ02000022 clade_85 N N Actinobacillus pleuropneumoniae 46 NR_074857clade_88 N N Actinobacillus ureae 48 AEVG01000167 clade_88 N NHaemophilus aegyptius 969 AFBC01000053 clade_88 N N Haemophilus ducreyi970 AE017143 clade_88 N OP Haemophilus haemolyticus 973 JN175335clade_88 N N Haemophilus influenzae 974 AADP01000001 clade_88 N OPHaemophilus parahaemolyticus 975 GU561425 clade_88 N N Haemophilusparainfluenzae 976 AEWU01000024 clade_88 N N Haemophilusparaphrophaemolyticus 977 M75076 clade_88 N N Haemophilus somnus 979NC_008309 clade_88 N N Haemophilus sp. 70334 980 HQ680854 clade_88 N NHaemophilus sp. HK445 981 FJ685624 clade_88 N N Haemophilus sp. oralclone ASCA07 982 AY923117 clade_88 N N Haemophilus sp. oral clone ASCG06983 AY923147 clade_88 N N Haemophilus sp. oral clone BJ021 984 AY005034clade_88 N N Haemophilus sp. oral clone BJ095 985 AY005033 clade_88 N NHaemophilus sp. oral taxon 851 987 AGRK01000004 clade_88 N N Haemophilussputorum 988 AFNK01000005 clade_88 N N Histophilus somni 1003 AF549387clade_88 N N Mannheimia haemolytica 1195 ACZX01000102 clade_88 N NPasteurella bettyae 1433 L06088 clade_88 N N Moellerella wisconsensis1253 JN175344 clade_89 N N Morganella morganii 1265 AJ301681 clade_89 NN Morganella sp. JB_T16 1266 AJ781005 clade_89 N N Proteus mirabilis1582 ACLE01000013 clade_89 N N Proteus penneri 1583 ABVP01000020clade_89 N N Proteus sp. HS7514 1584 DQ512963 clade_89 N N Proteusvulgaris 1585 AJ233425 clade_89 N N Oribacterium sinus 1374 ACKX1000142clade_90 N N Oribacterium sp. ACB1 1375 HM120210 clade_90 N NOribacterium sp. ACB7 1376 HM120211 clade_90 N N Oribacterium sp. CM121377 HQ616374 clade_90 N N Oribacterium sp. ICM51 1378 HQ616397 clade_90N N Oribacterium sp. OBRC12 1379 HQ616355 clade_90 N N Oribacterium sp.oral taxon 108 1382 AFIH01000001 clade_90 N N Actinobacillusactinomycetemcomitans 44 AY362885 clade_92 N N Actinobacillussuccinogenes 47 CP000746 clade_92 N N Aggregatibacteractinomycetemcomitans 112 CP001733 clade_92 N N Aggregatibacteraphrophilus 113 CP001607 clade_92 N N Aggregatibacter segnis 114AEPS01000017 clade_92 N N Averyella dalhousiensis 194 DQ481464 clade_92N N Bisgaard Taxon 368 AY683487 clade_92 N N Bisgaard Taxon 369 AY683489clade_92 N N Bisgaard Taxon 370 AY683491 clade_92 N N Bisgaard Taxon 371AY683492 clade_92 N N Buchnera aphidicola 440 NR_074609 clade_92 N NCedecea davisae 499 AF493976 clade_92 N N Citrobacter amalonaticus 517FR870441 clade_92 N N Citrobacter braakii 518 NR_028687 clade_92 N NCitrobacter farmeri 519 AF025371 clade_92 N N Citrobacter freundii 520NR_028894 clade_92 N N Citrobacter gillenii 521 AF025367 clade_92 N NCitrobacter koseri 522 NC_009792 clade_92 N N Citrobacter murliniae 523AF025369 clade_92 N N Citrobacter rodentium 524 NR_074903 clade_92 N NCitrobacter sedlakii 525 AF025364 clade_92 N N Citrobacter sp. 30_2 526ACDJ01000053 clade_92 N N Citrobacter sp. KMSI_3 527 GQ468398 clade_92 NN Citrobacter werkmanii 528 AF025373 clade_92 N N Citrobacter youngae529 ABWL02000011 clade_92 N N Cronobacter malonaticus 737 GU122174clade_92 N N Cronobacter sakazakii 738 NC_009778 clade_92 N NCronobacter turicensis 739 FN543093 clade_92 N N Enterobacter aerogenes786 AJ251468 clade_92 N N Enterobacter asburiae 787 NR_024640 clade_92 NN Enterobacter cancerogenus 788 Z96078 clade_92 N N Enterobacter cloacae789 FP929040 clade_92 N N Enterobacter cowanii 790 NR_025566 clade_92 NN Enterobacter hormaechei 791 AFHR01000079 clade_92 N N Enterobacter sp.247BMC 792 HQ122932 clade_92 N N Enterobacter sp. 638 793 NR_074777clade_92 N N Enterobacter sp. JC163 794 JN657217 clade_92 N NEnterobacter sp. SCSS 795 HM007811 clade_92 N N Enterobacter sp. TSE38796 HM156134 clade_92 N N Enterobacteriaceae bacterium 9_2_54FAA 797ADCU01000033 clade_92 N N Enterobacteriaceae bacterium CF01Ent_1 798AJ489826 clade_92 N N Enterobacteriaceae bacterium Smarlab 3302238 799AY538694 clade_92 N N Escherichia albertii 824 ABKX01000012 clade_92 N NEscherichia coli 825 NC_008563 clade_92 N Category-B Escherichiafergusonii 826 CU928158 clade_92 N N Escherichia hermannii 827 HQ407266clade_92 N N Escherichia sp. 1_1_43 828 ACID01000033 clade_92 N NEscherichia sp. 4_1_40B 829 ACDM02000056 clade_92 N N Escherichia sp. B4830 EU722735 clade_92 N N Escherichia vulneris 831 NR_041927 clade_92 NN Ewingella americana 877 JN175329 clade_92 N N Haemophilus genomosp. P2oral clone MB3_C24 971 DQ003621 clade_92 N N Haemophilus genomosp. P3oral clone MB3_C38 972 DQ003635 clade_92 N N Haemophilus sp. oral cloneJM053 986 AY349380 clade_92 N N Hafnia alvei 989 DQ412565 clade_92 N NKlebsiella oxytoca 1024 AY292871 clade_92 N OP Klebsiella pneumoniae1025 CP000647 clade_92 N OP Klebsiella sp. AS10 1026 HQ616362 clade_92 NN Klebsiella sp. Co9935 1027 DQ068764 clade_92 N N Klebsiella sp.enrichment culture clone SRC_DSD25 1036 HM195210 clade_92 N N Klebsiellasp. OBRC7 1028 HQ616353 clade_92 N N Klebsiella sp. SP_BA 1029 FJ999767clade_92 N N Klebsiella sp. SRC_DSD1 1033 GU797254 clade_92 N NKlebsiella sp. SRC_DSD11 1030 GU797263 clade_92 N N Klebsiella sp.SRC_DSD12 1031 GU797264 clade_92 N N Klebsiella sp. SRC_DSD15 1032GU797267 clade_92 N N Klebsiella sp. SRC_DSD2 1034 GU797253 clade_92 N NKlebsiella sp. SRC_DSD6 1035 GU797258 clade_92 N N Klebsiella variicola1037 CP001891 clade_92 N N Kluyvera ascorbata 1038 NR_028677 clade_92 NN Kluyvera cryocrescens 1039 NR_028803 clade_92 N N Leminorellagrimontii 1159 AJ233421 clade_92 N N Leminorella richardii 1160 HF558368clade_92 N N Pantoea agglomerans 1409 AY335552 clade_92 N N Pantoeaananatis 1410 CP001875 clade_92 N N Pantoea brenneri 1411 EU216735clade_92 N N Pantoea citrea 1412 EF688008 clade_92 N N Pantoea conspicua1413 EU216737 clade_92 N N Pantoea septica 1414 EU216734 clade_92 N NPasteurella dagmatis 1434 ACZR01000003 clade_92 N N Pasteurellamultocida 1435 NC_002663 clade_92 N N Plesiomonas shigelloides 1469X60418 clade_92 N N Raoultella ornithinolytica 1617 AB364958 clade_92 NN Raoultella planticola 1618 AF129443 clade_92 N N Raoultella terrigena1619 NR_037085 clade_92 N N Salmonella bongori 1683 NR_041699 clade_92 NCategory-B Salmonella enterica 1672 NC_011149 clade_92 N Category-BSalmonella enterica 1673 NC_011205 clade_92 N Category-B Salmonellaenterica 1674 DQ344532 clade_92 N Category-B Salmonella enterica 1675ABEH02000004 clade_92 N Category-B Salmonella enterica 1676 ABAK02000001clade_92 N Category-B Salmonella enterica 1677 NC_011080 clade_92 NCategory-B Salmonella enterica 1678 EU118094 clade_92 N Category-BSalmonella enterica 1679 NC_011094 clade_92 N Category-B Salmonellaenterica 1680 AE014613 clade_92 N Category-B Salmonella enterica 1682ABFH02000001 clade_92 N Category-B Salmonella enterica 1684 ABEM01000001clade_92 N Category-B Salmonella enterica 1685 ABAM02000001 clade_92 NCategory-B Salmonella typhimurium 1681 DQ344533 clade_92 N Category-BSalmonella typhimurium 1686 AF170176 clade_92 N Category-B Serratiafonticola 1718 NR_025339 clade_92 N N Serratia liquefaciens 1719NR_042062 clade_92 N N Serratia marcescens 1720 GU826157 clade_92 N NSerratia odorifera 1721 ADBY01000001 clade_92 N N Serratiaproteamaculans 1722 AAUN01000015 clade_92 N N Shigella boydii 1724AAKA01000007 clade_92 N Category-B Shigella dysenteriae 1725 NC_007606clade_92 N Category-B Shigella flexneri 1726 AE005674 clade_92 NCategory-B Shigella sonnei 1727 NC_007384 clade_92 N Category-BTatumella ptyseos 1916 NR_025342 clade_92 N N Trabulsiella guamensis1925 AYS73830 clade_92 N N Yersinia aldovae 2019 AJ871363 clade_92 N OPYersinia aleksiciae 2020 AJ627597 clade_92 N OP Yersinia bercovieri 2021AF366377 clade_92 N OP Yersinia enterocolitica 2022 FR729477 clade_92 NCategory-B Yersinia frederiksenii 2023 AF366379 clade_92 N OP Yersiniaintermedia 2024 AF366380 clade_92 N OP Yersinia kristensenii 2025ACCA01000078 clade_92 N OP Yersinia mollaretii 2026 NR_027546 clade_92 NOP Yersinia pestis 2027 AE013632 clade_92 N Category-A Yersiniapseudotuberculosis 2028 NC_009708 clade_92 N OP Yersinia rohdei 2029ACCD01000071 clade_92 N OP Yokenella regensburgei 2030 AB273739 clade_92N N Conchiformibius kuhniae 669 NR_041821 clade_94 N N Morococcuscerebrosus 1267 JN175352 clade_94 N N Neisseria bacilliformis 1328AFAY01000058 clade_94 N N Neisseria cinerea 1329 ACDY01000037 clade_94 NN Neisseria flavescens 1331 ACQV01000025 clade_94 N N Neisseriagonorrhoeae 1333 CP002440 clade_94 N OP Neisseria lactamica 1334ACEQ01000095 clade_94 N N Neisseria macacae 1335 AFQE01000146 clade_94 NN Neisseria meningitidis 1336 NC_003112 clade_94 N OP Neisseria mucosa1337 ACDX01000110 clade_94 N N Neisseria pharyngis 1338 AJ239281clade_94 N N Neisseria polysaccharea 1339 ADBE01000137 clade_94 N NNeisseria sicca 1340 ACKO02000016 clade_94 N N Neisseria sp. KEM232 1341GQ203291 clade_94 N N Neisseria sp. oral clone AP132 1344 AY005027clade_94 N N Neisseria sp. oral strain B33KA 1346 AY005028 clade_94 N NNeisseria sp. oral taxon 014 1347 ADEA01000039 clade_94 N N Neisseriasp. TM10_1 1343 DQ279352 clade_94 N N Neisseria subflava 1348ACEO01000067 clade_94 N N Okadaella gastrococcus 1365 HQ699465 clade_98N N Streptococcus agalactiae 1785 AAJO01000130 clade_98 N NStreptococcus alactolyticus 1786 NR_041781 clade_98 N N Streptococcusaustralis 1788 AEQR01000024 clade_98 N N Streptococcus bovis 1789AEEL01000030 clade_98 N N Streptococcus canis 1790 AJ413203 clade_98 N NStreptococcus constellatus 1791 AY277942 clade_98 N N Streptococcuscristatus 1792 AEVC01000028 clade_98 N N Streptococcus dysgalactiae 1794AP010935 clade_98 N N Streptococcus equi 1795 CP001129 clade_98 N NStreptococcus equinus 1796 AEVB01000043 clade_98 N N Streptococcusgallolyticus 1797 FR824043 clade_98 N N Streptococcus genomosp. C1 1798AY278629 clade_98 N N Streptococcus genomosp. C2 1799 AY278630 clade_98N N Streptococcus genomosp. C3 1800 AY278631 clade_98 N N Streptococcusgenomosp. C4 1801 AY278632 clade_98 N N Streptococcus genomosp. C5 1802AY278633 clade_98 N N Streptococcus genomosp. C6 1803 AY278634 clade_98N N Streptococcus genomosp. C7 1804 AY278635 clade_98 N N Streptococcusgenomosp. C8 1805 AY278609 clade_98 N N Streptococcus gordonii 1806NC_009785 clade_98 N N Streptococcus infantarius 1807 ABJK02000017clade_98 N N Streptococcus infantis 1808 AFNN01000024 clade_98 N NStreptococcus intermedius 1809 NR_028736 clade_98 N N Streptococcuslutetiensis 1810 NR_037096 clade_98 N N Streptococcus massiliensis 1811AY769997 clade_98 N N Streptococcus mitis 1813 AM157420 clade_98 N NStreptococcus oligofermentans 1815 AY099095 clade_98 N N Streptococcusoralis 1816 ADMV01000001 clade_98 N N Streptococcus parasanguinis 1817AEKM01000012 clade_98 N N Streptococcus pasteurianus 1818 AP012054clade_98 N N Streptococcus peroris 1819 AEVF01000016 clade_98 N NStreptococcus pneumoniae 1820 AE008537 clade_98 N N Streptococcusporcinus 1821 EF121439 clade_98 N N Streptococcus pseudopneumoniae 1822FJ827123 clade_98 N N Streptococcus pseudoporcinus 1823 AENS01000003clade_98 N N Streptococcus pyogenes 1824 AE006496 clade_98 N OPStreptococcus ratti 1825 X58304 clade_98 N N Streptococcus sanguinis1827 NR_074974 clade_98 N N Streptococcus sinensis 1828 AF432857clade_98 N N Streptococcus sp. 2_1_36FAA 1831 ACOI01000028 clade_98 N NStreptococcus sp. 2285_97 1830 AJ131965 clade_98 N N Streptococcus sp.ACS2 1834 HQ616360 clade_98 N N Streptococcus sp. AS20 1835 HQ616366clade_98 N N Streptococcus sp. BS35a 1836 HQ616369 clade_98 N NStreptococcus sp. C150 1837 ACRI01000045 clade_98 N N Streptococcus sp.CM6 1838 HQ616372 clade_98 N N Streptococcus sp. ICM10 1840 HQ616389clade_98 N N Streptococcus sp. ICM12 1841 HQ616390 clade_98 N NStreptococcus sp. ICM2 1842 HQ616386 clade_98 N N Streptococcus sp. ICM41844 HQ616387 clade_98 N N Streptococcus sp. ICM45 1843 HQ616394clade_98 N N Streptococcus sp. M143 1845 ACRK01000025 clade_98 N NStreptococcus sp. M334 1846 ACRL01000052 clade_98 N N Streptococcus sp.oral clone ASB02 1849 AY923121 clade_98 N N Streptococcus sp. oral cloneASCA03 1850 DQ272504 clade_98 N N Streptococcus sp. oral clone ASCA041851 AY923116 clade_98 N N Streptococcus sp. oral clone ASCA09 1852AY923119 clade_98 N N Streptococcus sp. oral clone ASCB04 1853 AY923123clade_98 N N Streptococcus sp. oral clone ASCB06 1854 AY923124 clade_98N N Streptococcus sp. oral clone ASCC04 1855 AY923127 clade_98 N NStreptococcus sp. oral clone ASCC05 1856 AY923128 clade_98 N NStreptococcus sp. oral clone ASCC12 1857 DQ272507 clade_98 N NStreptococcus sp. oral clone ASCD01 1858 AY923129 clade_98 N NStreptococcus sp. oral clone ASCD09 1859 AY923130 clade_98 N NStreptococcus sp. oral clone ASCD10 1860 DQ272509 clade_98 N NStreptococcus sp. oral clone ASCE03 1861 AY923134 clade_98 N NStreptococcus sp. oral clone ASCE04 1862 AY953253 clade_98 N NStreptococcus sp. oral clone ASCE05 1863 DQ272510 clade_98 N NStreptococcus sp. oral clone ASCE06 1864 AY923135 clade_98 N NStreptococcus sp. oral clone ASCE09 1865 AY923136 clade_98 N NStreptococcus sp. oral clone ASCE10 1866 AY923137 clade_98 N NStreptococcus sp. oral clone ASCE12 1867 AY923138 clade_98 N NStreptococcus sp. oral clone ASCF05 1868 AY923140 clade_98 N NStreptococcus sp. oral clone ASCF07 1869 AY953255 clade_98 N NStreptococcus sp. oral clone ASCF09 1870 AY923142 clade_98 N NStreptococcus sp. oral clone ASCG04 1871 AY923145 clade_98 N NStreptococcus sp. oral clone BW009 1872 AY005042 clade_98 N NStreptococcus sp. oral clone CH016 1873 AY005044 clade_98 N NStreptococcus sp. oral clone GK051 1874 AY349413 clade_98 N NStreptococcus sp. oral clone GM006 1875 AY349414 clade_98 N NStreptococcus sp. oral clone P2PA_41 P2 1876 AY207051 clade_98 N NStreptococcus sp. oral clone P4PA_30 P4 1877 AY207064 clade_98 N NStreptococcus sp. oral taxon 071 1878 AEEP01000019 clade_98 N NStreptococcus sp. oral taxon G59 1879 GU432132 clade_98 N NStreptococcus sp. oral taxon G62 1880 GU432146 clade_98 N NStreptococcus sp. oral taxon G63 1881 GU432150 clade_98 N NStreptococcus suis 1882 FM252032 clade_98 N N Streptococcus thermophilus1883 CP000419 clade_98 N N Streptococcus salivarius 1826 AGBV01000001clade_98 N N Streptococcus uberis 1884 HQ391900 clade_98 N NStreptococcus urinalis 1885 DQ303194 clade_98 N N Streptococcusvestibularis 1886 AEKO01000008 clade_98 N N Streptococcus viridans 1887AF076036 clade_98 N N Synergistetes bacterium oral clone 03 5 D05 1908GU227192 clade_98 N N

TABLE 1A Exemplary Immunomodulatory Bacterial Species   Alkaliphilusmetalliredigens Ammonifex degensii Anaerofustis stercorihominisAnaerostipes caccae Anaerotruncus colihominis Bacillus amyloliquefaciensBacillus anthracis Bacillus cellulosilyticus Bacillus cereus Bacillusclausii Bacillus coagulans Bacillus cytotoxicus Bacillus haloduransBacillus licheniformis Bacillus pumilus Bacillus subtilis Bacillusthuringiensis Bacillus weihenstephanensis Blautia (Ruminococcus)hansenii Blautia (Ruminococcus) obeum Brevibacillus brevis Bryantellaformatexigens Caldicellulosiruptor saccharolyticus CandidatusDesulforudis audaxviator Carboxydibrachium pacificum Carboxydothermushydrogenoformans Clostridium acetobutylicum Clostridium asparagiformeClostridium bartlettii Clostridium beuerinckii Clostridium bolteaeClostridium botulinum A str. ATCC 19397 Clostridium botulinum B str.Eklund 17B Clostridium butyricum pathogenic E4 str. BoNT BL5262Clostridium carboxidivorans Clostridium cellulolyticum Clostridiumcellulovorans Clostridium difficile Clostridium (Hungatella) hathewayiClostridium hylemonae Clostridium kluyveri Clostridium leptumClostridium methylpentosum Clostridium (Tyzzerella) nexile Clostridiumnovyi NT Clostridium papyrosolvens Clostridium perfringens Clostridiumphytofermentans ISDg Clostridium scindens Clostridium sp. 7_2_43FAAClostridium sporogenes Clostridium tetani Clostridium thermocellumCoprococcus comes Desulfotomaculum reducens Dorea longicatenaEubacterium eligens Eubacterium hallii Eubacterium rectale Eubacteriumventriosum Faecalibacterium prausnitzii Geobacillus kaustophilusGeobacillus sp. G11MC16 Geobacillus thermodenitrificans Heliobacteriummodesticaldum Lysinibacillus sphaericus Oceanobacillus iheyensisPaenibacillus sp. JDR-2 Pelotomaculum thermopropionicum Roseburiaintestinalis Ruminococcus bromii Ruminococcus gnavus Ruminococcustorques Subdoligranulum variabile Symbiobacterium thermophilumThermoanaerobacter italicus Thermoanaerobacter tengcongensisThermoanaerobacterium thermosaccharolyticum Thermosinus carboxydivorans

TABLE 1B Exemplary Bacteria Useful in the Present InventionAcidaminococcus intestini Acinetobacter baumannii Acinetobacter lwoffiiAkkermansia muciniphila Alistipes putredinis Alistipes shahiiAnaerostipes hadrus Anaerotruncus colihominis Bacteroides caccaeBacteroides cellulosilyticus Bacteroides dorei Bacteroides eggerthiiBacteroides finegoldii Bacteroides fragilis Bacteroides massiliensisBacteroides ovatus Bacteroides salanitronis Bacteroides salyersiaeBacteroides sp. 1_1_6 Bacteroides sp. 3_1_23 Bacteroides sp. D20Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides vulgatusBifidobacterium adolescentis Bifidobacterium bifidum Bifidobacteriumbreve Bifidobacterium faecale Bifidobacterium kashiwanohenseBifidobacterium longum subsp. longum Bifidobacterium pseudocatenulatumBifidobacterium stercoris Blautia (Ruminococcus) coccoides Blautiafaecis Blautia glucerasea Blautia (Ruminococcus) hansenii Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus) Blautia(Ruminococcus) luti Blautia (Ruminococcus) obeum Blautia producta(Ruminococcus productus) Blautia (Ruminococcus) schinkii Blautiastercoris Blautia uncultured bacterium clone BLKE_a03_2 (GenBank:EU469501.1) Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1) Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1) Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1) Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2) Blautiawexlerae Candidatus Arthromitus sp. SFB-mouse-Yit Catenibacteriummitsuokai Clostridiaceae bacterium (Dielma fastidiosa) JC13Clostridiales bacterium 1_7_47FAA Clostridium asparagiforme Clostridiumbolteae Clostridium clostridioforme Clostridium glycyrrhizinilyticumClostridium (Hungatella) hathewayi Clostridium histolyticum Clostridiumindolis Clostridium leptum Clostridium (Tyzzerella) nexile Clostridiumperfringens Clostridium (Erysipelatoclostridium) ramosum Clostridiumscindens Clostridium sp. 14774 Clostridium sp. 7_3_54FAA Clostridium sp.HGF2 Clostridium symbiosum Collinsella aerofaciens Collinsellaintestinalis Coprobacillus sp. D7 Coprococcus catus Coprococcus comesDorea formicigenerans Dorea longicatena Enterococcus faecalisEnterococcus faecium Erysipelotrichaceae bacterium 3_1_53 Escherichiacoli Escherichia coli S88 Eubacterium eligens Eubacterium fissicatenaEubacterium ramulus Eubacterium rectale Faecalibacterium prausnitziiFlavonifractor plautii Fusobacterium mortiferum Fusobacterium nucleatumHoldemania filiformis Hydrogenoanaerobacterium saccharovorans Klebsiellaoxytoca Lachnospiraceae bacterium 3_1_57FAA_CT1 Lachnospiraceaebacterium 7_1_58FAA Lachnospiraceae bacterium 5_1_57FAA Lactobacilluscasei Lactobacillus rhamnosus Lactobacillus ruminis Lactococcus caseiOdoribacter splanchnicus Oscillibacter valericigenes Parabacteroidesgordonii Parabacteroides johnsonii Parabacteroides merdae Pediococcusacidilactici Peptostreptococcus asaccharolyticus Propionibacteriumgranulosum Roseburia intestinalis Roseburia inulinivorans Ruminococcusfaecis Ruminococcus gnavus Ruminococcus sp. ID8 Ruminococcus torquesSlackia piriformis Staphylococcus epidermidis Staphylococcussaprophyticus Streptococcus cristatus Streptococcus dysgalactiae subsp.equisimilis Streptococcus infantis Streptococcus oralis Streptococcussanguinis Streptococcus viridans Streptococcus thermophilus Veillonelladispar

TABLE 1C Exemplary Bacteria Useful in the Present Invention  Anaerotruncus colihominis strain 13 Blautia producta strain 6Clostridium bolteae strain 7 Clostridiaceae bacterium JC13 strain 8Clostridiales bacterium 1_7_47FAA strain 28 Clostridium sp. 7_3_54FAAstrain 16 Clostridium asparagiforme strain 15 Clostridiumclostridioforme Clostridium (Hungatella) hathewayi strain 4 Clostridiumindolis strain 9 Clostridium (Erysipelatoclostridium) ramosum strain 18Clostridium scindens strain 26 Clostridium sp. 14774 strain 1Eubacterium fissicatena strain 21 Hydrogenoanaerobacteriumsaccharovorans Lachnospiraceae bacterium 3_1_57FAA strain 27Lachnospiraceae bacterium 3_1_57FAA strain 29 Lachnospiraceae bacterium7_1_58FAA strain 3 Oscillibacter valericigenes Ruminococcus sp. ID8strain 14

TABLE 1D Exemplary Bacteria Useful in the Present Invention  Bacteroides caccae Bacteroides eggerthii Bacteroides ovatus Bacteroidessp. 1_1_6 Bacteroides sp. 3_1_23 Bacteroides sp. D20 Bacteroidesvulgatus Bifidobacterium adolescentis Bifidobacterium pseudocatenulatumBlautia (Ruminococcus) obeum Blautia producta (Ruminococcus productus)Blautia (Ruminococcus) schinkii Clostridium (Hungatella) hathewayiClostridium (Tyzzerella) nexile Clostridium sp. HGF2 Clostridiumsymbiosum Collinsella aerofaciens Coprobacillus sp. D7 Coprococcus catusCoprococcus comes Dorea formicigenerans Dorea longicatena Enterococcusfaecalis Erysipelotrichaceae bacterium 3_1_53 Escherichia coliEscherichia coli S88 Eubacterium eligens Eubacterium rectaleFaecalibacterium prausnitzii Lachnospiraceae bacterium 5_1_57FAAOdoribacter splanchnicus Parabacteroides merdae Roseburia intestinalisRuminococcus torques Streptococcus thermophilus

TABLE 1E Exemplary Bacteria Useful in the Present Invention  Akkermansia muciniphila Enterococcus faecalis Klebsiella oxytocaLactobacillus rhamnosus Staphylococcus epidermidis Streptococcusviridans Veillonella dispar

TABLE 1F Exemplary Bacteria Useful in the Present Invention  Acinetobacter baumannii Acinetobacter lwoffii Akkermansia muciniphilaAlistipes shahii Anaerotruncus colihominis Bacteroides caccaeBacteroides dorei Bacteroides eggerthii Bacteroides finegoldiiBacteroides fragilis Bacteroides massiliensis Bacteroides ovatusBacteroides salanitronis Bacteroides sp. 1_1_6 Bacteroides sp. 3_1_23Bacteroides sp. D20 Bacteroides thetaiotaomicron Bacteroides uniformisBacteroides vulgatus Bifidobacterium adolescentis Bifidobacterium breveBifidobacterium pseudocatenulatum Blautia (Ruminococcus) coccoidesBlautia faecis Blautia glucerasea Blautia (Ruminococcus) hanseniiBlautia hydrogenotrophica (Ruminococcus hydrogenotrophicus) Blautia(Ruminococcus) luti Blautia (Ruminococcus) obeum Blautia producta(Ruminococcus productus) Blautia (Ruminococcus) schinkii Blautiastercoris Blautia wexlerae Candidatus Arthromitus sp. SFB-mouse-YitClostridiaceae bacterium (Dielma fastidiosa) JC13 Clostridialesbacterium 1_7_47FAA Clostridium asparagiforme Clostridium bolteaeClostridium clostridioforme Clostridium (Hungatella) hathewayiClostridium histolyticum Clostridium indolis Clostridium leptumClostridium (Tyzzerella) nexile Clostridium perfringens Clostridium(Erysipelatoclostridium) ramosum Clostridium scindens Clostridium sp.14774 Clostridium sp. 7_3_54FAA Clostridium sp. HGF2 Clostridiumsymbiosum Collinsella aerofaciens Coprobacillus sp. D7 Coprococcus catusCoprococcus comes Dorea formicigenerans Dorea longicatena Enterococcusfaecium Erysipelotrichaceae bacterium 3_1_53 Escherichia coliEscherichia coli S88 Eubacterium eligens Eubacterium fissicatenaEubacterium rectale Faecalibacterium prausnitzii Fusobacteriummortiferum Fusobacterium nucleatum Hydrogenoanaerobacteriumsaccharovorans Lachnospiraceae bacterium 3_1_57FAA_CT1 Lachnospiraceaebacterium 7_1_58FAA Lachnospiraceae bacterium 5_1_57FAA Lactobacilluscasei Lactococcus casei Odoribacter splanchnicus Oscillibactervalericigenes Parabacteroides johnsonii Parabacteroides merdaePediococcus acidilactici Peptostreptococcus asaccharolyticusPropionibacterium granulosum Roseburia intestinalis Ruminococcus gnavusRuminococcus sp. ID8 Ruminococcus torques Staphylococcus saprophyticusStreptococcus thermophilus

TABLE 2A Species identified as germinable and sporulatable by colonypicking GAM + Sweet B + Sweet OTU BBA FOS/inulin M2GSC FOS/Inulin GAMTotal Blautia producta 1 1 Clostridium bartlettii 4 1 5 Clostridiumbolteae 2 5 1 8 Clostridium botulinum 5 5 Clostridium butyricum 37 43 81 33 122 Clostridium celatum 4 1 5 Clostridium clostridioforme 1 1 2Clostridium disporicum 26 26 22 33 50 157 Clostridium glycolicum 4 9 1427 Clostridium mayombei 2 2 4 Clostridium paraputrificum 8 8 33 16 6 71Clostridium sordellii 14 14 Clostridium sp. 7_2_43FAA 1 1 Clostridiumsymbiosum 3 3 Clostridium tertium 1 1 2 (blank) 2 31 33 Totals 92 92 9292 92 460

TABLE 2B Species identified as germinable by 16S colony pick approach  Clostridium_paraputrificum Clostridium_disporicum Clostridium_glycolicumClostridium_bartlettii Clostridium_butyricum Ruminococcus_bromiiLachnospiraceae_bacterium_2_1_58FAA Eubacterium_hadrumTuricibacter_sanguinis Lachnospiraceae_bacterium_oral_taxon_F15Clostridium_perfringens Clostridium_bifermentans Roseburia_sp_11SE37Clostridium_quinii Ruminococcus_lactaris Clostridium_botulinumClostridium_tyrobutyricum Blautia_hansenii Clostridium_kluyveriClostridium_sp_JC122 Clostridium_hylemonae Clostridium_celatumClostridium_straminisolvens Clostridium_orbiscindens Roseburia_cecicolaEubacterium_tenue Clostridium_sp_7_2_43FAALachnospiraceae_bacterium_4_1_37FAA Eubacterium_rectaleClostridium_viride Ruminococcus_sp_K_1 Clostridium_symbiosumRuminococcus_torques Clostridium_algidicarnis

TABLE 2C Species identified as sporulatable by 16S NGS approach  Clostridium_paraputrificum Clostridium_bartlettiiLachnospiraceae_bacterium_2_1_58FAA Clostridium_disporicumRuminococcus_bromii Eubacterium_hadrum Clostridium_butyricumRoseburia_sp_11SE37 Clostridium_perfringens Clostridium_glycolicumClostridium_hylemonae Clostridium_orbiscindens Ruminococcus_lactarisClostridium_symbiosum Lachnospiraceae_bacterium_oral_taxon_F15Blautia_hansenii Turicibacter_sanguinis Clostridium_straminisolvensClostridium_botulinum Lachnospiraceae_bacterium_4_1_37FAARoseburia_cecicola Ruminococcus_sp_K_1 Clostridium_bifermentansEubacterium_rectale Clostridium_quinii Clostridium_virideClostridium_kluyveri Clostridium_tyrobutyricum Oscillibacter_sp_G2Clostridium_sp_JC122 Lachnospiraceae_bacterium_3_1_57FAAClostridium_aldenense Ruminococcus_torques Clostridium_sp_7_2_43FAAClostridium_celatum Eubacterium_sp_WAL_14571 Eubacterium_tenueLachnospiraceae_bacterium_5_1_57FAA Clostridium_clostridioformeClostridium_sp_YIT_12070 Blautia_sp_M25 Anaerostipes_caccaeRoseburia_inulinivorans Clostridium_sp_D5 Clostridium_asparagiformeCoprobacillus_sp_D7 Clostridium_sp_HGF2 Clostridium_citroniaeClostridium_difficile Oscillibacter_valericigenesClostridium_algidicarnis

TABLE 3 Criteria for stages of acute GVHD ORGAN Skin <25% 25-50% >50% orGeneralized (maculopapular rash generalised erythroderma with extent, %of body erythroderma bullous formation surface area) or desquamationLiver 34-50 51-102 103-255 >255 (bilirubin mmol L) [2-3] [3-6] [6-15][>15] [bilirubin mg/dL] ø) (or AST 150-750 U/L) Gut >30 mL kg or >60mL/kg or >90 mL/kg or >2000 mL/day or (daily diarrhea >500 mL (2) >1000mL >1500 mL severe abdominal volume) pain with or without ileus

TABLE 4 COGs for Identifying Prebiotic Activities COG COG ID COG NAMECATEGORY COG0022 Pyruvate/2-oxoglutarate/acetoin dehydrogenase complex,C dehydrogenase (E1) component COG0039 Malate/lactate dehydrogenase CCOG0045 Succinyl-CoA synthetase, beta subunit C COG0055 FoF1-type ATPsynthase, beta subunit C COG0056 FoF1-type ATP synthase, alpha subunit CCOG0074 Succinyl-CoA synthetase, alpha subunit C COG0114 Fumaratehydratase class II C COG0224 FoF1-type ATP synthase, gamma subunit CCOG0240 Glycerol-3-phosphate dehydrogenase C COG0243 Anaerobicselenocysteine-containing dehydrogenase C COG0247 Fe—S oxidoreductase CCOG0277 FAD/FMN-containing dehydrogenase C COG0280 PhosphotransacetylaseC COG0281 Malic enzyme C COG0282 Acetate kinase C COG0348 PolyferredoxinC COG0355 FoF1-type ATP synthase, epsilon subunit C COG0356 FoF1-typeATP synthase, membrane subunit a C COG0371 Glycerol dehydrogenase orrelated enzyme, iron-containing C ADH family COG0372 Citrate synthase CCOG0374 Ni,Fe-hydrogenase I large subunit C COG0377 NADH: ubiquinoneoxidoreductase 20 kD subunit (chhain B) or C related Fe—S oxidoreductaseCOG0426 Flavorubredoxin C COG0427 Acyl-CoA hydrolase C COG0431NAD(P)H-dependent FMN reductase C COG0435 Glutathionyl-hydroquinonereductase C COG0437 Fe—S-cluster-containing dehydrogenase component CCOG0479 Succinate dehydrogenase/fumarate reductase, Fe—S protein Csubunit COG0508 Pyruvate/2-oxoglutarate dehydrogenase complex, Cdihydrolipoamide acyltransferase (E2) component COG0538 Isocitratedehydrogenase C COG0546 Phosphoglycolate phosphatase, HAD superfamily CCOG0554 Glycerol kinase C COG0567 2-oxoglutarate dehydrogenase complex,dehydrogenase (E1) C component, and related enzymes COG0578Glycerol-3-phosphate dehydrogenase C COG0633 Ferredoxin C COG0636FoF1-type ATP synthase, membrane subunit C c/Archaeal/vacuolar-typeH+-ATPase, subunit K COG0644 Dehydrogenase (flavoprotein) C COG0649NADH: ubiquinone oxidoreductase 49 kD subunit (chain D) C COG0650Formate hydrogenlyase subunit 4 C COG0655 Multimeric flavodoxin WrbA CCOG0674 Pyruvate: ferredoxin oxidoreductase or related 2- C oxoacid:ferredoxin oxidoreductase, alpha subunit COG0680 Ni,Fe-hydrogenasematuration factor C COG0711 FoF1-type ATP synthase, membrane subunit bor b′ C COG0712 FoF1-type ATP synthase, delta subunit C COG0713 NADH:ubiquinone oxidoreductase subunit 11 or 4L (chain K) C COG0716Flavodoxin C COG0723 Rieske Fe—S protein C COG0778 Nitroreductase CCOG0838 NADH: ubiquinone oxidoreductase subunit 3 (chain A) C COG0839NADH: ubiquinone oxidoreductase subunit 6 (chain J) C COG0843Heme/copper-type cytochrome/quinol oxidase, subunit 1 C COG0852 NADH:ubiquinone oxidoreductase 27 kD subunit (chain C) C COG1005 NADH:ubiquinone oxidoreductase subunit 1 (chain H) C COG1007 NADH: ubiquinoneoxidoreductase subunit 2 (chain N) C COG1008 NADH: ubiquinoneoxidoreductase subunit 4 (chain M) C COG1012 Acyl-CoA reductase or otherNAD-dependent aldehyde C dehydrogenase COG1013 Pyruvate: ferredoxinoxidoreductase or related 2- C oxoacid: ferredoxin oxidoreductase, betasubunit COG1014 Pyruvate: ferredoxin oxidoreductase or related 2- Coxoacid: ferredoxin oxidoreductase, gamma subunit COG1017Hemoglobin-like flavoprotein C COG1018 Ferredoxin-NADP reductase CCOG1029 Formylmethanofuran dehydrogenase subunit B C COG1034 NADHdehydrogenase/NADH: ubiquinone oxidoreductase 75 C kD subunit (chain G)COG1035 Coenzyme F420-reducing hydrogenase, beta subunit C COG1036Archaeal flavoprotein C COG1038 Pyruvate carboxylase C COG1042 Acyl-CoAsynthetase (NDP forming) C COG1048 Aconitase A C COG1049 Aconitase B CCOG1053 Succinate dehydrogenase/fumarate reductase, flavoprotein Csubunit COG1071 TPP-dependent pyruvate or acetoin dehydrogenase subunitalpha C COG1139 L-lactate utilization protein LutB, contains aferredoxin-type C domain COG1141 Ferredoxin C COG1142Fe—S-cluster-containing hydrogenase component 2 C COG1143 Formatehydrogenlyase subunit 6/NADH: ubiquinone C oxidoreductase 23 kD subunit(chain I) COG1144 Pyruvate: ferredoxin oxidoreductase or related 2- Coxoacid: ferredoxin oxidoreductase, delta subunit COG1145 Ferredoxin CCOG1148 Heterodisulfide reductase, subunit A (polyferredoxin) C COG1150Heterodisulfide reductase, subunit C C COG1152 COdehydrogenase/acetyl-CoA synthase alpha subunit C COG1153Formylmethanofuran dehydrogenase subunit D C COG1155Archaeal/vacuolar-type H+-ATPase catalytic subunit A/Vma1 C COG1156Archaeal/vacuolar-type H+-ATPase subunit B/Vma2 C COG1182 FMN-dependentNADH-azoreductase C COG1229 Formylmethanofuran dehydrogenase subunit A CCOG1249 Pyruvate/2-oxoglutarate dehydrogenase complex, Cdihydrolipoamide dehydrogenase (E3) component or related enzyme COG1251NAD(P)H-nitrite reductase, large subunit C COG1252 NADH dehydrogenase,FAD-containing subunit C COG1254 Acylphosphatase C COG1269Archaeal/vacuolar-type H+-ATPase subunit I/STV1 C COG1271 Cytochromebd-type quinol oxidase, subunit 1 C COG1274 Phosphoenolpyruvatecarboxykinase, GTP-dependent C COG1282 NAD/NADP transhydrogenase betasubunit C COG1290 Cytochrome b subunit of the bc complex C COG1294Cytochrome bd-type quinol oxidase, subunit 2 C COG1301Na+/H+-dicarboxylate symporter C COG1319 CO or xanthine dehydrogenase,FAD-binding subunit C COG1347 Na+-transporting NADH: ubiquinoneoxidoreductase, subunit C NqrD COG1359 Quinol monooxygenase YgiN CCOG1390 Archaeal/vacuolar-type H+-ATPase subunit E/Vma4 C COG1394Archaeal/vacuolar-type H+-ATPase subunit D/Vma8 C COG1436Archaeal/vacuolar-type H+-ATPase subunit F/Vma7 C COG1454 Alcoholdehydrogenase, class IV C COG1456 CO dehydrogenase/acetyl-CoA synthasegamma subunit C (corrinoid Fe—S protein) COG1526 Formate dehydrogenaseassembly factor FdhD C COG1527 Archaeal/vacuolar-type H+-ATPase subunitC/Vma6 C COG1529 CO or xanthine dehydrogenase, Mo-binding subunit CCOG1556 L-lactate utilization protein LutC, contains LUD domain CCOG1584 Succinate-acetate transporter protein C COG1592 Rubrerythrin CCOG1614 CO dehydrogenase/acetyl-CoA synthase beta subunit C COG1620L-lactate permease C COG1622 Heme/copper-type cytochrome/quinol oxidase,subunit 2 C COG1625 Fe—S oxidoreductase, related to NifB/MoaA family CCOG1679 Predicted aconitase C COG1726 Na+-transporting NADH: ubiquinoneoxidoreductase, subunit C NqrA COG1740 Ni,Fe-hydrogenase I small subunitC COG1757 Na+/H+ antiporter NhaC C COG1773 Rubredoxin C COG1795Formaldehyde-activating enzyme nesessary for methanogenesis C COG1805Na+-transporting NADH: ubiquinone oxidoreductase, subunit C NqrB COG1838Tartrate dehydratase beta subunit/Fumarate hydratase class I, C- Cterminal domain COG1845 Heme/copper-type cytochrome/quinol oxidase,subunit 3 C COG1853 NADH-FMN oxidoreductase RutF, flavin reductase C(DIM6/NTAB) family COG1866 Phosphoenolpyruvate carboxykinase,ATP-dependent C COG1880 CO dehydrogenase/acetyl-CoA synthase epsilonsubunit C COG1882 Pyruvate-formate lyase C COG1883 Na+-transportingmethylmalonyl-CoA/oxaloacetate C decarboxylase, beta subunit COG1894NADH: ubiquinone oxidoreductase, NADH-binding 51 kD C subunit (chain F)COG1902 2,4-dienoyl-CoA reductase or related NADH-dependent C reductase,Old Yellow Enzyme (OYE) family COG1905 NADH: ubiquinone oxidoreductase24 kD subunit (chain E) C COG1908 Coenzyme F420-reducing hydrogenase,delta subunit C COG1927 F420-dependent methylenetetrahydromethanopterinC dehydrogenase COG1941 Coenzyme F420-reducing hydrogenase, gammasubunit C COG1951 Tartrate dehydratase alpha subunit/Fumarate hydrataseclass I, C N-terminal domain COG1969 Ni,Fe-hydrogenase I cytochrome bsubunit C COG1979 Alcohol dehydrogenase YqhD, Fe-dependent ADH family CCOG2009 Succinate dehydrogenase/fumarate reductase, cytochrome b Csubunit COG2010 Cytochrome c, mono-and diheme variants C COG2025Electron transfer flavoprotein, alpha subunit C COG2033Desulfoferrodoxin, superoxide reductase-like (SORL) domain C COG2037Formylmethanofuran: tetrahydromethanopterin formyltransferase C COG2041Periplasmic DMSO/TMAO reductase YedYZ, molybdopterin- C dependentcatalytic subunit COG2048 Heterodisulfide reductase, subunit B C COG2055Malate/lactate/ureidoglycolate dehydrogenase, LDH2 family C COG2069 COdehydrogenase/acetyl-CoA synthase delta subunit (corrinoid C Fe—Sprotein) COG2080 Aerobic-type carbon monoxide dehydrogenase, smallsubunit, C CoxS/CutS family COG2086 Electron transfer flavoprotein,alpha and beta subunits C COG2142 Succinate dehydrogenase, hydrophobicanchor subunit C COG2191 Formylmethanofuran dehydrogenase subunit E CCOG2209 Na+-transporting NADH: ubiquinone oxidoreductase, subunit C NqrECOG2210 Peroxiredoxin family protein C COG2218 Formylmethanofurandehydrogenase subunit C C COG2224 Isocitrate lyase C COG2225 Malatesynthase C COG2326 Polyphosphate kinase 2, PPK2 family C COG2352Phosphoenolpyruvate carboxylase C COG2414 Aldehyde: ferredoxinoxidoreductase C COG2421 Acetamidase/formamidase C COG2440Ferredoxin-like protein FixX C COG2609 Pyruvate dehydrogenase complex,dehydrogenase (E1) C component COG2717 Periplasmic DMSO/TMAO reductaseYedYZ, heme-binding C membrane subunit COG2811 Archaeal/vacuolar-typeH+-ATPase subunit H C COG2828 2-Methylaconitate cis-trans-isomerase PrpF(2-methyl citrate C pathway) COG2838 Monomeric isocitrate dehydrogenaseC COG2851 Mg2+/citrate symporter C COG2857 Cytochrome c1 C COG2863Cytochrome c553 C COG2864 Cytochrome b subunit of formate dehydrogenaseC COG2869 Na+-transporting NADH: ubiquinone oxidoreductase, subunit CNqrC COG2871 Na+-transporting NADH: ubiquinone oxidoreductase, subunit CNqrF COG2878 Na+-translocating ferredoxin: NAD+ oxidoreductase RNF, RnfBC subunit COG2993 Cbb3-type cytochrome oxidase, cytochrome c subunit CCOG3005 Tetraheme cytochrome c subunit of nitrate or TMAO reductase CCOG3029 Fumarate reductase subunit C C COG3038 Cytochrome b561 C COG3051Citrate lyase, alpha subunit C COG3052 Citrate lyase, gamma subunit CCOG3053 Citrate lyase synthetase C COG3069 C4-dicarboxylate transporterC COG3080 Fumarate reductase subunit D C COG3125 Heme/copper-typecytochrome/quinol oxidase, subunit 4 C COG3181 Tripartite-typetricarboxylate transporter, receptor component C TctC COG3202 ATP/ADPtranslocase C COG3241 Azurin C COG3245 Cytochrome c5 C COG3258Cytochrome c C COG3259 Coenzyme F420-reducing hydrogenase, alpha subunitC COG3260 Ni,Fe-hydrogenase III small subunit C COG3261Ni,Fe-hydrogenase III large subunit C COG3262 Ni,Fe-hydrogenase IIIcomponent G C COG3278 Cbb3-type cytochrome oxidase, subunit 1 C COG3288NAD/NADP transhydrogenase alpha subunit C COG3302 DMSO reductase anchorsubunit C COG3312 FoF1-type ATP synthase assembly protein I C COG3411(2Fe—2S) ferredoxin C COG3426 Butyrate kinase C COG3427 Carbon monoxidedehydrogenase subunit G C COG3474 Cytochrome c2 C COG3493 Na+/citrate orNa+/malate symporter C COG3630 Na+-transportingmethylmalonyl-CoA/oxaloacetate C decarboxylase, gamma subunit COG3658Cytochrome b C COG3761 NADH: ubiquinone oxidoreductase 17.2 kD subunit CCOG3783 Soluble cytochrome b562 C COG3794 Plastocyanin C COG3808 Na+ orH+-translocating membrane pyrophosphatase C COG3909 Cytochrome c556 CCOG3978 Acetolactate synthase small subunit, contains ACT domain CCOG4036 Energy-converting hydrogenase Eha subunit G C COG4037Energy-converting hydrogenase Eha subunit F C COG4038 Energy-convertinghydrogenase Eha subunit E C COG4039 Energy-converting hydrogenase Ehasubunit C C COG4041 Energy-converting hydrogenase Eha subunit B CCOG4042 Energy-converting hydrogenase Eha subunit A C COG40745,10-methenyltetrahydromethanopterin hydrogenase C COG4078Energy-converting hydrogenase Eha subunit H C COG4106 Trans-aconitatemethyltransferase C COG4147 Na+(or H+)/acetate symporter ActP C COG4221NADP-dependent 3-hydroxy acid dehydrogenase YdfG C COG4231 TPP-dependentindolepyruvate ferredoxin oxidoreductase, alpha C subunit COG4237Hydrogenase-4 membrane subunit HyfE C COG4459 Periplasmic nitratereductase system, NapE component C COG4624 Iron only hydrogenase largesubunit, C-terminal domain C COG4654 Cytochrome c551/c552 C COG4656Na+-translocating ferredoxin: NAD+ oxidoreductase RNF, RnfC C subunitCOG4657 Na+-translocating ferredoxin: NAD+ oxidoreductase RNF, RnfA Csubunit COG4658 Na+-translocating ferredoxin: NAD+ oxidoreductase RNF,RnfD C subunit COG4659 Na+-translocating ferredoxin: NAD+ oxidoreductaseRNF, RnfG C subunit COG4660 Na+-translocating ferredoxin: NAD+oxidoreductase RNF, RnfE C subunit COG4736 Cbb3-type cytochrome oxidase,subunit 3 C COG4802 Ferredoxin-thioredoxin reductase, catalytic subunitC COG5012 Methanogenic corrinoid protein MtbC1 C COG5016Pyruvate/oxaloacetate carboxyltransferase C COG0021 Transketolase GCOG0033 Phosphoglucomutase G COG0036 Pentose-5-phosphate-3-epimerase GCOG0057 Glyceraldehyde-3-phosphate dehydrogenase/erythrose-4- Gphosphate dehydrogenase COG0058 Glucan phosphorylase G COG0120 Ribose5-phosphate isomerase G COG0126 3-phosphoglycerate kinase G COG0148Enolase G COG0149 Triosephosphate isomerase G COG0153 Galactokinase GCOG0158 Fructose-1,6-bisphosphatase G COG0166 Glucose-6-phosphateisomerase G COG0176 Transaldolase G COG0191 Fructose/tagatosebisphosphate aldolase G COG0205 6-phosphofructokinase G COG0235Ribulose-5-phosphate 4-epimerase/Fuculose-1-phosphate G aldolase COG0246Mannitol-1-phosphate/altronate dehydrogenases G COG02693-keto-L-gulonate-6-phosphate decarboxylase G COG0279 Phosphoheptoseisomerase G COG0296 1,4-alpha-glucan branching enzyme G COG0297 Glycogensynthase G COG0362 6-phosphogluconate dehydrogenase G COG03636-phosphogluconolactonase/Glucosamine-6-phosphate G isomerase/deaminaseCOG0364 Glucose-6-phosphate 1-dehydrogenase G COG0366 Glycosidase GCOG0380 Trehalose-6-phosphate synthase G COG0383 Alpha-mannosidase GCOG0395 ABC-type glycerol-3-phosphate transport system, permease Gcomponent COG0406 Broad specificity phosphatase PhoE G COG0448ADP-glucose pyrophosphorylase G COG0469 Pyruvate kinase G COG0471 Di-andtricarboxylate transporter G COG0483 Archaealfructose-1,6-bisphosphatase or related enzyme of G inositolmonophosphatase family COG0524 Sugar or nucleoside kinase, ribokinasefamily G COG0574 Phosphoenolpyruvate synthase/pyruvate phosphatedikinase G COG0579 L-2-hydroxyglutarate oxidase LhgO G COG0580 Glyceroluptake facilitator and related aquaporins (Major G Intrinsic ProteinFamily) COG0588 Phosphoglycerate mutase (BPG-dependent) G COG0662Mannose-6-phosphate isomerase, cupin superfamily G COG0676D-hexose-6-phosphate mutarotase G COG0696 Phosphoglycerate mutase(BPG-independent, AlkP superfamily) G COG0698 Ribose 5-phosphateisomerase RpiB G COG0738 Fucose permease G COG08002-keto-3-deoxy-6-phosphogluconate aldolase G COG0837 Glucokinase GCOG1015 Phosphopentomutase G COG1023 6-phosphogluconate dehydrogenase(decarboxylating) G COG1064 D-arabinose 1-dehydrogenase, Zn-dependentalcohol G dehydrogenase family COG1069 Ribulose kinase G COG1070 Sugar(pentulose or hexulose) kinase G COG1080 Phosphoenolpyruvate-proteinkinase (PTS system EI component G in bacteria) COG1082 Sugar phosphateisomerase/epimerase G COG1085 Galactose-1-phosphate uridylyltransferaseG COG1105 Fructose-1-phosphate kinase or kinase (PfkB) G COG1109Phosphomannomutase G COG1129 ABC-type sugar transport system, ATPasecomponent G COG1172 Ribose/xylose/arabinose/galactoside ABC-typetransport system, G permease component COG1175 ABC-type sugar transportsystem, permease component G COG1216 Glycosyltransferase, GT2 family GCOG1263 Phosphotransferase system IIC components, glucose/maltose/N- Gacetylglucosamine-specific COG1264 Phosphotransferase system IIBcomponents G COG1299 Phosphotransferase system, fructose-specific IICcomponent G COG1312 D-mannonate dehydratase G COG1440 Phosphotransferasesystem cellobiose-specific component IIB G COG1445 Phosphotransferasesystem fructose-specific component IIB G COG1447 Phosphotransferasesystem cellobiose-specific component IIA G COG1449Alpha-amylase/alpha-mannosidase, GH57 family G COG1455Phosphotransferase system cellobiose-specific component IIC G COG1472Periplasmic beta-glucosidase and related glycosidases G COG1482Mannose-6-phosphate isomerase, class I G COG1486Alpha-galactosidase/6-phospho-beta-glucosidase, family 4 of G glycosylhydrolase COG1494 Fructose-1,6-bisphosphatase/sedoheptulose1,7-bisphosphatase G or related protein COG1501 Alpha-glucosidase,glycosyl hydrolase family GH31 G COG1523 Pullulanase/glycogendebranching enzyme G COG1543 Predicted glycosyl hydrolase, contains GH57and DUF1957 G domains COG1554 Trehalose and maltose hydrolase (possiblephosphorylase) G COG1593 TRAP-type C4-dicarboxylate transport system,large permease G component COG1621 Sucrose-6-phosphate hydrolase SacC,GH32 family G COG1626 Neutral trehalase G COG1638 TRAP-typeC4-dicarboxylate transport system, periplasmic G component COG16404-alpha-glucanotransferase G COG1653 ABC-type glycerol-3-phosphatetransport system, periplasmic G component COG1803 Methylglyoxal synthaseG COG1819 UDP: flavonoid glycosyltransferase YjiC, YdhE family G COG1820N-acetylglucosamine-6-phosphate deacetylase G COG1830Fructose-bisphosphate aldolase class Ia, DhnA family G COG1850 Ribulose1,5-bisphosphate carboxylase, large subunit, or a G RuBisCO-like proteinCOG1869 D-ribose pyranose/furanose isomerase RbsD G COG1874Beta-galactosidase GanA G COG1877 Trehalose-6-phosphatase G COG1879ABC-type sugar transport system, periplasmic component, G containsN-terminal xre family HTH domain COG1892 Phosphoenolpyruvate carboxylaseG COG1904 Glucuronate isomerase G COG1929 Glycerate kinase G COG1980Archaeal fructose 1,6-bisphosphatase G COG2017 Galactose mutarotase orrelated enzyme G COG2074 2-phosphoglycerate kinase G COG20792-methylcitrate dehydratase PrpD G COG2115 Xylose isomerase G COG2120N-acetylglucosaminyl deacetylase, LmbE family G COG2133Glucose/arabinose dehydrogenase, beta-propeller fold G COG2140 Oxalatedecarboxylase/archaeal phosphoglucose isomerase, G cupin superfamilyCOG2152 Predicted glycosyl hydrolase, GH43/DUF377 family G COG2160L-arabinose isomerase G COG2182 Maltose-binding periplasmic protein MalEG COG2190 Phosphotransferase system IIA component G COG2211Na+/melibiose symporter or related transporter G COG2213Phosphotransferase system, mannitol-specific IIBC component G COG2220L-ascorbate metabolism protein UlaG, beta-lactamase G superfamilyCOG2271 Sugar phosphate permease G COG2273 Beta-glucanase, GH16 family GCOG2301 Citrate lyase beta subunit G COG2342 Endo alpha-1,4polygalactosaminidase, GH114 family (was G erroneously annotated asCys-tRNA synthetase) COG2376 Dihydroxyacetone kinase G COG2379Glycerate-2-kinase G COG2407 L-fucose isomerase or related protein GCOG2513 2-Methylisocitrate lyase and related enzymes, PEP mutase Gfamily COG2704 Anaerobic C4-dicarboxylate transporter G COG27066-phosphogluconolactonase, cycloisomerase 2 family G COG2721 Altronatedehydratase G COG2723Beta-glucosidase/6-phospho-beta-glucosidase/beta-galactosidase G COG2730Aryl-phospho-beta-D-glucosidase BglC, GH1 family G COG2731Beta-galactosidase, beta subunit G COG2814 Predicted arabinose effluxpermease, MFS family G COG2861 Uncharacterized conserved protein YibQ,putative G polysaccharide deacetylase 2 family COG2893Phosphotransferase system, mannose/fructose-specific G component IIACOG2942 Mannose or cellobiose epimerase, N-acyl-D-glucosamine 2- Gepimerase family COG2971 BadF-type ATPase, related to humanN-acetylglucosamine G kinase COG3001 Fructosamine-3-kinase G COG3010Putative N-acetylmannosamine-6-phosphate epimerase G COG3037Ascorbate-specific PTS system EIIC-type component UlaA G COG3090TRAP-type C4-dicarboxylate transport system, small permease G componentCOG3250 Beta-galactosidase/beta-glucuronidase G COG3265 Gluconate kinaseG COG3280 Maltooligosyltrehalose synthase G COG3281 Predicted trehalosesynthase G COG3325 Chitinase, GH18 family G COG3345 Alpha-galactosidaseG COG3347 Rhamnose utilisation protein RhaD, predicted bifunctional Galdolase and dehydrogenase COG3386 Sugar lactone lactonase YvrE GCOG3387 Glucoamylase (glucan-1,4-alpha-glucosidase), GH15 family GCOG3405 Endo-1,4-beta-D-glucanase Y G COG3408 Glycogen debranchingenzyme (alpha-1,6-glucosidase) G COG3414 Phosphotransferase system,galactitol-specific IIB component G COG3429 Glucose-6-phosphatedehydrogenase assembly protein OpcA, G contains a peptidoglycan-bindingdomain COG3444 Phosphotransferase system, mannose/fructose/N- Gacetylgalactosamine-specific component IIB COG3459 Cellobiosephosphorylase G COG3469 Chitinase G COG3507 Beta-xylosidase G COG3525N-acetyl-beta-hexosaminidase G COG3534 Alpha-L-arabinofuranosidase GCOG3537 Putative alpha-1,2-mannosidase G COG3588 Fructose-bisphosphatealdolase class 1 G COG3594 Fucose 4-O-acetylase or relatedacetyltransferase G COG3622 Hydroxypyruvate isomerase G COG3623L-ribulose-5-phosphate 3-epimerase UlaE G COG36352,3-bisphosphoglycerate-independent phosphoglycerate mutase, G archealtype COG3661 Alpha-glucuronidase G COG3664 Beta-xylosidase G COG3669Alpha-L-fucosidase G COG3684 Tagatose-1,6-bisphosphate aldolase GCOG3693 Endo-1,4-beta-xylanase, GH35 family G COG3709 Ribose1,5-bisphosphokinase PhnN G COG3715 Phosphotransferase system,mannose/fructose/N- G acetylgalactosamine-specific component IIC COG3716Phosphotransferase system, mannose/fructose/N- Gacetylgalactosamine-specific component IID COG3717 5-keto 4-deoxyuronateisomerase G COG3718 5-deoxy-D-glucuronate isomerase G COG3730Phosphotransferase system sorbitol-specific component IIC G COG3731Phosphotransferase system sorbitol-specific component IIA G COG3732Phosphotransferase system sorbitol-specific component IIBC G COG37342-keto-3-deoxy-galactonokinase G COG3769 Predictedmannosyl-3-phosphoglycerate phosphatase, HAD G superfamily COG3775Phosphotransferase system, galactitol-specific IIC component G COG3822D-lyxose ketol-isomerase G COG3833 ABC-type maltose transport system,permease component G COG3836 2-keto-3-deoxy-L-rhamnonate aldolase RhmA GCOG3839 ABC-type sugar transport system, ATPase component G COG3855Fructose-1,6-bisphosphatase G COG3866 Pectate lyase G COG3867Arabinogalactan endo-1,4-beta-galactosidase G COG3892 Myo-inositolcatabolism protein IolC G COG3934 Endo-1,4-beta-mannosidase G COG3936Membrane-bound acyltransferase YfiQ, involved in biofilm G formationCOG3940 Beta-xylosidase, GH43 family G COG3954 Phosphoribulokinase GCOG3957 Phosphoketolase G COG3958 Transketolase, C-terminal subunit GCOG3959 Transketolase, N-terminal subunit G COG3962 TPP-dependenttrihydroxycyclohexane-1,2-dione (THcHDO) G dehydratase, myo-inositolmetabolism COG3979 Chitodextrinase G COG4124 Beta-mannanase G COG4130Predicted sugar epimerase, xylose isomerase-like family G COG4154L-fucose mutarotase/ribose pyranase, RbsD/FucU family G COG4193Beta-N-acetylglucosaminidase G COG4209 ABC-type polysaccharide transportsystem, permease G component COG4211 ABC-type glucose/galactosetransport system, permease G component COG4213 ABC-type xylose transportsystem, periplasmic component G COG4214 ABC-type xylose transportsystem, permease component G COG4225 Rhamnogalacturonyl hydrolase YesR GCOG4284 UDP-N-acetylglucosamine pyrophosphorylase G COG4451 Ribulosebisphosphate carboxylase small subunit G COG4468 Galactose-1-phosphateuridylyltransferase G COG4573 Tagatose-1,6-bisphosphate aldolasenon-catalytic subunit G AgaZ/GatZ COG4580 Maltoporin (phage lambda andmaltose receptor) G COG4632 Exopolysaccharide biosynthesis proteinrelated to N- G acetylglucosamine-1-phosphodiester alpha-N-acety . . .COG4668 Mannitol/fructose-specific phosphotransferase system, IIA Gdomain COG4724 Endo-beta-N-acetylglucosaminidase D G COG4806 L-rhamnoseisomerase G COG4809 Archaeal ADP-dependentphosphofructokinase/glucokinase G COG4813 Trehalose utilization proteinG COG4833 Predicted alpha-1,6-mannanase, GH76 family G COG4975 Glucoseuptake protein GlcU G COG4993 Glucose dehydrogenase G COG5017UDP-N-acetylglucosamine transferase subunit ALG13 G COG5026 Hexokinase GCOG5263 Glucan-binding domain (YG repeat) G COG0002N-acetyl-gamma-glutamylphosphate reductase E COG0006 Xaa-Proaminopeptidase E COG0010 Arginase family enzyme E COG0014 Gamma-glutamylphosphate reductase E COG0019 Diaminopimelate decarboxylase E COG0031Cysteine synthase E COG0040 ATP phosphoribosyltransferase E COG0065Homoaconitase/3-isopropylmalate dehydratase large subunit E COG00663-isopropylmalate dehydratase small subunit E COG0067 Glutamate synthasedomain 1 E COG0069 Glutamate synthase domain 2 E COG0070 Glutamatesynthase domain 3 E COG0076 Glutamate or tyrosine decarboxylase or arelated PLP-dependent E protein COG0077 Prephenate dehydratase E COG0078Ornithine carbamoyltransferase E COG0079 Histidinol-phosphate/aromaticaminotransferase or cobyric acid E decarboxylase COG0082 Chorismatesynthase E COG0083 Homoserine kinase E COG0106Phosphoribosylformimino-5-aminoimidazole carboxamide E ribonucleotide(ProFAR) isomerase COG0107 Imidazole glycerol phosphate synthase subunitHisF E COG0112 Glycine/serine hydroxymethyltransferase E COG0118Imidazoleglycerol phosphate synthase glutamine E amidotransferasesubunit HisH COG0119 Isopropylmalate/homocitrate/citramalate synthases ECOG0128 5-enolpyruvylshikimate-3-phosphate synthase E COG0131Imidazoleglycerol phosphate dehydratase HisB E COG0133 Tryptophansynthase beta chain E COG0134 Indole-3-glycerol phosphate synthase ECOG0135 Phosphoribosylanthranilate isomerase E COG0136Aspartate-semialdehyde dehydrogenase E COG0137 Argininosuccinatesynthase E COG0139 Phosphoribosyl-AMP cyclohydrolase E COG0140Phosphoribosyl-ATP pyrophosphohydrolase E COG0141 Histidinoldehydrogenase E COG0159 Tryptophan synthase alpha chain E COG01604-aminobutyrate aminotransferase or related aminotransferase E COG0165Argininosuccinate lyase E COG0169 Shikimate 5-dehydrogenase E COG0174Glutamine synthetase E COG0182 Methylthioribose-1-phosphate isomerase(methionine salvage E pathway), a paralog of eIF-2B alpha subunitCOG0241 Histidinol phosphatase or a related phosphatase E COG0253Diaminopimelate epimerase E COG0260 Leucyl aminopeptidase E COG0263Glutamate 5-kinase E COG0287 Prephenate dehydrogenase E COG0289Dihydrodipicolinate reductase E COG0308 Aminopeptidase N E COG0334Glutamate dehydrogenase/leucine dehydrogenase E COG0337 3-dehydroquinatesynthetase E COG0339 Zn-dependent oligopeptidase E COG0345Pyrroline-5-carboxylate reductase E COG0367 Asparagine synthetase B(glutamine-hydrolyzing) E COG0403 Glycine cleavage system protein P(pyridoxal-binding), N- E terminal domain COG0404 Glycine cleavagesystem T protein (aminomethyltransferase) E COG0405Gamma-glutamyltranspeptidase E COG0410 ABC-type branched-chain aminoacid transport system, ATPase E component COG0411 ABC-typebranched-chain amino acid transport system, ATPase E component COG0421Spermidine synthase E COG0436 Aspartate/methionine/tyrosineaminotransferase E COG0440 Acetolactate synthase, small subunit ECOG0460 Homoserine dehydrogenase E COG0498 Threonine synthase E COG0506Proline dehydrogenase E COG0509 Glycine cleavage system H protein(lipoate-binding) E COG0520 Selenocysteine lyase/Cysteine desulfurase ECOG0527 Aspartokinase E COG0531 Amino acid transporter E COG0547Anthranilate phosphoribosyltransferase E COG0548 Acetylglutamate kinaseE COG0549 Carbamate kinase E COG0559 Branched-chain amino acid ABC-typetransport system, E permease component COG0560 Phosphoserine phosphataseE COG0591 Na+/proline symporter E COG0620 Methionine synthase II(cobalamin-independent) E COG0624 Acetylornithinedeacetylase/Succinyl-diaminopimelate E desuccinylase or relateddeacylase COG0626 Cystathionine beta-lyase/cystathionine gamma-synthaseE COG0646 Methionine synthase I (cobalamin-dependent), methyltransferaseE domain COG0665 Glycine/D-amino acid oxidase (deaminating) E COG0683ABC-type branched-chain amino acid transport system, E periplasmiccomponent COG0685 5,10-methylenetetrahydrofolate reductase E COG0686Alanine dehydrogenase E COG0687 Spermidine/putrescine-bindingperiplasmic protein E COG0703 Shikimate kinase E COG0709 Selenophosphatesynthase E COG0710 3-dehydroquinate dehydratase E COG07223-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase E COG0747ABC-type transport system, periplasmic component E COG0754Glutathionylspermidine synthase E COG0757 3-dehydroquinate dehydratase ECOG0765 ABC-type amino acid transport system, permease component ECOG0786 Na+/glutamate symporter E COG0804 Urease alpha subunit E COG0814Amino acid permease E COG0831 Urease gamma subunit E COG0832 Urease betasubunit E COG0833 Amino acid permease E COG1003 Glycine cleavage systemprotein P (pyridoxal-binding), C- E terminal domain COG1027 Aspartateammonia-lyase E COG1045 Serine acetyltransferase E COG1104 Cysteinesulfinate desulfinase/cysteine desulfurase or related E enzyme COG1113L-asparagine transporter and related permeases E COG1114 Branched-chainamino acid permeases E COG1115 Na+/alanine symporter E COG1125 ABC-typeproline/glycine betaine transport system, ATPase E component COG1126ABC-type polar amino acid transport system, ATPase E component COG1135ABC-type methionine transport system, ATPase component E COG1164Oligoendopeptidase F E COG1166 Arginine decarboxylase (spermidinebiosynthesis) E COG1171 Threonine dehydratase E COG1174 ABC-typeproline/glycine betaine transport system, permease E component COG1176ABC-type spermidine/putrescine transport system, permease E component ICOG1177 ABC-type spermidine/putrescine transport system, permease Ecomponent II COG1231 Monoamine oxidase E COG1246 N-acetylglutamatesynthase or related acetyltransferase, GNAT E family COG1247 L-aminoacid N-acyltransferase YncA E COG1279 Arginine exporter protein ArgO ECOG1280 Threonine/homoserine/homoserine lactone efflux protein E COG1296Predicted branched-chain amino acid permease (azaleucine E resistance)COG1350 Predicted alternative tryptophan synthase beta-subunit (paralogE of TrpB) COG1362 Aspartyl aminopeptidase E COG1364 N-acetylglutamatesynthase (N-acetylornithine E aminotransferase) COG1410 Methioninesynthase I, cobalamin-binding domain E COG1446 Isoaspartyl peptidase orL-asparaginase, Ntn-hydrolase E superfamily COG1448Aspartate/tyrosine/aromatic aminotransferase E COG1465 3-dehydroquinatesynthase, class II E COG1505 Prolyl oligopeptidase PreP, S9A serinepeptidase family E COG1506 Dipeptidyl aminopeptidase/acylaminoacylpeptidase E COG1509 L-lysine 2,3-aminomutase (EF-P beta-lysylationpathway) E COG1586 S-adenosylmethionine decarboxylase or argininedecarboxylase E COG1605 Chorismate mutase E COG1685 Archaeal shikimatekinase E COG1687 Branched-chain amino acid transport protein Az1D ECOG1748 Saccharopine dehydrogenase, NADP-dependent E COG1760 L-serinedeaminase E COG1770 Protease II E COG1791 Acireductone dioxygenase(methionine salvage), cupin E superfamily COG1823 L-cystine uptakeprotein TcyP, sodium: dicarboxylate symporter E family COG1834N-Dimethylarginine dimethylaminohydrolase E COG1878 Kynurenineformamidase E COG1897 Homoserine trans-succinylase E COG1945Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) E COG1982Arginine/lysine/ornithine decarboxylase E COG1984 Allophanate hydrolasesubunit 2 E COG2008 Threonine aldolase E COG2011 ABC-type methioninetransport system, permease component E COG2021 Homoserineacetyltransferase E COG2040 Homocysteine/selenocysteine methylase(S-methylmethionine- E dependent) COG2049 Allophanate hydrolase subunit1 E COG2056 Predicted histidine transporter YuiF, NhaC family E COG2066Glutaminase E COG2071 Gamma-glutamyl-gamma-aminobutyrate hydrolase PuuDE (putrescine degradation), contains GATase1-like domain COG2095 Smallneutral amino acid transporter SnatA, MarC family E COG2113 ABC-typeproline/glycine betaine transport system, periplasmic E componentCOG2171 Tetrahydrodipicolinate N-succinyltransferase E COG2195 Di- ortripeptidase E COG2235 Arginine deiminase E COG2309 Leucylaminopeptidase (aminopeptidase T) E COG2317 Zn-dependentcarboxypeptidase, M32 family E COG2355 Zn-dependent dipeptidase,microsomal dipeptidase homolog E COG2362 D-aminopeptidase E COG2423Ornithine cyclodeaminase/archaeal alanine dehydrogenase, mu- Ecrystallin family COG2502 Asparagine synthetase A E COG25151-aminocyclopropane-1-carboxylate deaminase/D-cysteine E desulfhydrase,PLP-dependent ACC family COG2716 Glycine cleavage system regulatoryprotein E COG2755 Lysophospholipase L1 or related esterase E COG2856Zn-dependent peptidase ImmA, M78 family E COG2873O-acetylhomoserine/O-acetylserine sulfhydrylase, pyridoxal Ephosphate-dependent COG2876 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase E COG2902 NAD-specific glutamate dehydrogenase E COG2939Carboxypeptidase C (cathepsin A) E COG2957 Agmatine/peptidylargininedeiminase E COG2981 Uncharacterized protein involved in cysteinebiosynthesis E COG2986 Histidine ammonia-lyase E COG2987 Urocanatehydratase E COG2988 Succinylglutamate desuccinylase E COG3033Tryptophanase E COG3048 D-serine dehydratase E COG3075 Anaerobicglycerol-3-phosphate dehydrogenase E COG3104 Dipeptide/tripeptidepermease E COG3138 Arginine/ornithine N-succinyltransferase beta subunitE COG3186 Phenylalanine-4-hydroxylase E COG3192 Ethanolamine transporterEutH, required for ethanolamine E utilization at low pH COG32003-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) E synthase, class IICOG3232 5-carboxymethyl-2-hydroxymuconate isomerase E COG3340 PeptidaseE E COG3404 Formiminotetrahydrofolate cyclodeaminase E COG3457 Predictedamino acid racemase E COG3483 Tryptophan 2,3-dioxygenase (vermilion) ECOG3579 Aminopeptidase C E COG3591 V8-like Glu-specific endopeptidase ECOG3616 D-serine deaminase, pyridoxal phosphate-dependent E COG3633Na+/serine symporter E COG3643 Glutamate formiminotransferase E COG3681L-cysteine desulfidase E COG3705 ATP phosphoribosyltransferaseregulatory subunit HisZ E COG3724 Succinylarginine dihydrolase E COG3741N-formylglutamate amidohydrolase E COG3799 Methylaspartate ammonia-lyaseE COG3842 ABC-type Fe3+/spermidine/putrescine transport systems, EATPase components COG3844 Kynureninase E COG3931 PredictedN-formylglutamate amidohydrolase E COG3938 Proline racemase E COG3968Glutamine synthetase type III E COG3977 Alanine-alpha-ketoisovalerate(or valine-pyruvate) E aminotransferase COG4091 Predicted homoserinedehydrogenase, contains C-terminal SAF E domain COG4126Asp/Glu/hydantoin racemase E COG4160 ABC-type arginine/histidinetransport system, permease E component COG4161 ABC-type argininetransport system, ATPase component E COG4166 ABC-type oligopeptidetransport system, periplasmic component E COG4175 ABC-typeproline/glycine betaine transport system, ATPase E component COG4176ABC-type proline/glycine betaine transport system, permease E componentCOG4177 ABC-type branched-chain amino acid transport system, E permeasecomponent COG4187 Arginine utilization protein RocB E COG4215 ABC-typearginine transport system, permease component E COG4229Enolase-phosphatase E1 involved in merthionine salvage E COG4230 Delta1-pyrroline-5-carboxylate dehydrogenase E COG4302 Ethanolamineammonia-lyase, small subunit E COG4303 Ethanolamine ammonia-lyase, largesubunit E COG4311 Sarcosine oxidase delta subunit E COG43592-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate E phosphatase(methionine salvage) COG4392 Branched-chain amino acid transport proteinE COG4401 Chorismate mutase E COG4413 Urea transporter E COG4448L-asparaginase II E COG4583 Sarcosine oxidase gamma subunit E COG4597ABC-type amino acid transport system, permease component E COG4598ABC-type histidine transport system, ATPase component E COG4608 ABC-typeoligopeptide transport system, ATPase component E COG4690 Dipeptidase ECOG4766 Ethanolamine utilization protein EutQ, cupin superfamily E(function unknown) COG4810 Ethanolamine utilization protein EutS,ethanolamine utilization E microcompartment shell protein COG4812Ethanolamine utilization cobalamin adenosyltransferase E COG4816Ethanolamine utilization protein EutL, ethanolamine utilization Emicrocompartment shell protein COG4819 Ethanolamine utilization proteinEutA, possible chaperonin E protecting lyase from inhibition COG4820Ethanolamine utilization protein EutJ, possible chaperonin E COG48575-Methylthioribose kinase, methionine salvage pathway E COG4865Glutamate mutase epsilon subunit E COG4917 Ethanolamine utilizationprotein EutP, contains a P-loop NTPase E domain COG4992Acetylornithine/succinyldiaminopimelate/putrescine E aminotransferaseCOG5006 Threonine/homoserine efflux transporter RhtA E COG0020Undecaprenyl pyrophosphate synthase I COG0183 Acetyl-CoAacetyltransferase I COG0204 1-acyl-sn-glycerol-3-phosphateacyltransferase I COG0245 2C-methyl-D-erythritol 2,4-cyclodiphosphatesynthase I COG0331 Malonyl CoA-acyl carrier protein transacylase ICOG0332 3-oxoacyl-[acyl-carrier-protein] synthase III I COG0344Phospholipid biosynthesis protein PlsY, probable glycerol-3- I phosphateacyltransferase COG0365 Acyl-coenzyme A synthetase/AMP-(fatty) acidligase I COG0416 Fatty acid/phospholipid biosynthesis enzyme I COG0439Biotin carboxylase I COG0446 NADPH-dependent 2,4-dienoyl-CoA reductase,sulfur reductase, I or a related oxidoreductase COG0558Phosphatidylglycerophosphate synthase I COG0575 CDP-diglyceridesynthetase I COG0584 Glycerophosphoryl diester phosphodiesterase ICOG0623 Enoyl-[acyl-carrier-protein] reductase (NADH) I COG0657 Acetylesterase/lipase I COG0671 Membrane-associated phospholipid phosphatase ICOG0688 Phosphatidylserine decarboxylase I COG0736 Phosphopantetheinyltransferase (holo-ACP synthase) I COG0743 1-deoxy-D-xylulose 5-phosphatereductoisomerase I COG0761 4-Hydroxy-3-methylbut-2-enyl diphosphatereductase IspH I COG0764 3-hydroxymyristoyl/3-hydroxydecanoyl-(acylcarrier protein) I dehydratase COG0777 Acetyl-CoA carboxylase betasubunit I COG0818 Diacylglycerol kinase I COG08214-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase I IspG/GcpE COG0824Acyl-CoA thioesterase FadM I COG0825 Acetyl-CoA carboxylase alphasubunit I COG1022 Long-chain acyl-CoA synthetase (AMP-forming) I COG1024Enoyl-CoA hydratase/carnithine racemase I COG1075 Triacylglycerolesterase/lipase EstA, alpha/beta hydrolase fold I COG1133 ABC-typelong-chain fatty acid transport system, fused I permease and ATPasecomponents COG1183 Phosphatidylserine synthase I COG12112-C-methyl-D-erythritol 4-phosphate cytidylyltransferase I COG1213Choline kinase I COG1250 3-hydroxyacyl-CoA dehydrogenase I COG1257Hydroxymethylglutaryl-CoA reductase I COG1260 Myo-inositol-1-phosphatesynthase I COG1267 Phosphatidylglycerophosphatase A I COG1307 Fattyacid-binding protein DegV (function unknown) I COG1398 Fatty-aciddesaturase I COG1443 Isopentenyldiphosphate isomerase I COG1502Phosphatidylserine/phosphatidylglycerophosphate/cardiolipin I synthaseor related enzyme COG1560 Lauroyl/myristoyl acyltransferase I COG1562Phytoene/squalene synthetase I COG1577 Mevalonate kinase I COG1607Acyl-CoA hydrolase I COG1608 Isopentenyl phosphate kinase I COG1646Heptaprenylglyceryl phosphate synthase I COG1657 Squalene cyclase ICOG1788 Acyl CoA: acetate/3-ketoacid CoA transferase, alpha subunit ICOG1804 Crotonobetainyl-CoA: carnitine CoA-transferase CaiB and Irelated acyl-CoA transferases COG1884 Methylmalonyl-Co A mutase,N-terminal domain/subunit I COG1924 Activator of 2-hydroxyglutaryl-CoAdehydratase (HSP70-class I ATPase domain) COG1946 Acyl-CoA thioesteraseI COG1947 4-diphosphocytidyl-2C-methyl-D-erythritol kinase I COG1960Acyl-CoA dehydrogenase related to the alkylation response I protein AidBCOG1968 Undecaprenyl pyrophosphate phosphatase UppP I COG2030 Acyldehydratase I COG2031 Short chain fatty acids transporter I COG2057 AcylCoA: acetate/3-ketoacid CoA transferase, beta subunit I COG2067Long-chain fatty acid transport protein I COG2084 3-hydroxyisobutyratedehydrogenase or related beta- I hydroxyacid dehydrogenase COG2134CDP-diacylglycerol pyrophosphatase I COG2185 Methylmalonyl-CoA mutase,C-terminal domain/subunit I (cobalamin-binding) COG2230 Cyclopropanefatty-acyl-phospholipid synthase and related I methyltransferasesCOG2246 Putative flippase GtrA (transmembrane translocase of Ibactoprenol-linked glucose) COG2267 Lysophospholipase, alpha-betahydrolase superfamily I COG2272 Carboxylesterase type B I COG2854ABC-type transporter Mla maintaining outer membrane lipid I asymmetry,periplasmic MlaC component COG2930 Lipid-binding SYLF domain I COG2937Glycerol-3-phosphate O-acyltransferase I COG3000 Steroldesaturase/sphingolipid hydroxylase, fatty acid I hydroxylasesuperfamily COG3007 Trans-2-enoyl-CoA reductase I COG3124 Acyl carrierprotein phosphodiesterase I COG3154 Predicted lipid carrier proteinYhbT, SCP2 domain I COG3239 Fatty acid desaturase I COG3243Poly(3-hydroxyalkanoate) synthetase I COG3255 Putative sterol carrierprotein I COG3356 Predicted membrane-associated lipid hydrolase, neutralI ceramidase superfamily COG3407 Mevalonate pyrophosphate decarboxylaseI COG3425 3-hydroxy-3-methylglutaryl CoA synthase I COG3475Phosphorylcholine metabolism protein LicD I COG3675 Predicted lipase ICOG3777 Hydroxyacyl-ACP dehydratase HTD2, hotdog domain I COG3882Predicted enzyme involved in methoxymalonyl-ACP I biosynthesis COG3884Acyl-ACP thioesterase I COG3963 Phospholipid N-methyltransferase ICOG4247 3-phytase (myo-inositol-hexaphosphate 3-phosphohydrolase) ICOG4281 Acyl-CoA-binding protein I COG4395 Predicted lipid-bindingtransport protein, Tim44 family I COG4553 Poly-beta-hydroxyalkanoatedepolymerase I COG4667 Predicted phospholipase, patatin/cPLA2 family ICOG4670 Acyl CoA: acetate/3-ketoacid CoA transferase I COG4706 Predicted3-hydroxylacyl-ACP dehydratase, HotDog domain I COG4770Acetyl/propionyl-CoA carboxylase, alpha subunit I COG4781Membrane-anchored glycerophosphoryl diester I phosphodiesterase(GDPDase), membrane domain COG4799 Acetyl-CoA carboxylase,carboxyltransferase component I COG4850 Phosphatidate phosphatase APP1 ICOG4981 Enoyl reductase domain of yeast-type FAS1 I COG49823-oxoacyl-ACP reductase domain of yeast-type FAS1 I COG5083Phosphatidate phosphatase PAH1, contains Lipin and LNS2 I domains, canbe involved in plasmid maintenance COG0473 Isocitrate/isopropylmalatedehydrogenase CE COG0604 NADPH: quinone reductase or relatedZn-dependent CR oxidoreductase COG0129 Dihydroxyaciddehydratase/phosphogluconate dehydratase EG COG1363 Putativeaminopeptidase FrvX EG COG0493 NADPH-dependent glutamate synthase betachain or related ER oxidoreductase COG1063 Threonine dehydrogenase orrelated Zn-dependent ER dehydrogenase COG1168 Bifunctional PLP-dependentenzyme with beta-cystathionase ER and maltose regulon repressoractivities COG1387 Histidinol phosphatase or related hydrolase of thePHP family ER COG3185 4-hydroxyphenylpyruvate dioxygenase and relatedhemolysins ER COG0347 Nitrogen regulatory protein PII TE COG0834ABC-type amino acid transport/signal transduction system, ET periplasmiccomponent/domain COG4677 Pectin methylesterase and related acyl-CoAthioesterases GI COG0637 Beta-phosphoglucomutase or related phosphatase,HAD GR superfamily COG2610 H+/gluconate symporter or related permease GRCOG1489 DNA-binding protein, stimulates sugar fermentation GT COG1762Phosphotransferase system mannitol/fructose-specific IIA GT domain(Ntr-type) COG1925 Phosphotransferase system, HPr and relatedphosphotransfer TG proteins COG3925 N-terminal domain of thephosphotransferase system fructose- GT specific component IIB COG4945Carbohydrate-binding DOMON domain GT COG1597 Diacylglycerol kinasefamily enzyme IR COG2303 Choline dehydrogenase or related flavoproteinIR COG3240 Phospholipase/lecithinase/hemolysin IR

TABLE 5 List of species enriched in alive GHVD patients: Lactobacillusgasseri Lactobacillus fermentum Lactobacillus reuteri Enterococcusfaecalis Enterococcus durans Enterococcus villorum Lactobacillusplantarum Pediococcus acidilactici Staphylococcus pasteuriStaphylococcus cohnii Streptococcus sanguinis Streptococcus sinensisStreptococcus mitis Streptococcus sp. SCA22 Streptococcus sp. CR-3145Streptococcus anginosus Streptococcus mutans Coprobacillus cateniformisClostridium saccharogumia Eubacterium dolichum DSM 3991 Clostridium sp.PPf35E6 Clostridium sordelli ATCC 9714 Ruminococcus torques Ruminococcusgnavus Clostridium clostridioforme Ruminococcus obeum Blautia productaClostridium sp. ID5 Megasphaera micronuciformis Veillonella parvulaClostridium methylpentosum Clostridium islandicum Faecalibacteriumprausnitzii Bacteroides uniformmis Bacteroides thetaiotaomicronBacteroides acidifaciens Bacteroides ovatus Bacteroides fragilisParabacteroides distasonis Propinionibacteirum propionicum Actinomycshyovaginalis Rothia mucilaginosa Rothia aeria Bifidobacterium breveScardovia inopinata Eggerthella lenta

TABLE 6 Anaerobic bacterial species tested for carbon source usage(Biolog plates) Species purchased: Species Freshly Isolated: R. gnavus(EPV1) Blautia luti BlnIX (EPV114) E. rectale (EPV2) Blautia luti ELU(EPV54) B. luti (EPV3) Ruminococcus gnavus (EPV102) B. wexlerae (EPV5)Blautia faecis (EPV78) C. leptum (EPV6) Ruminococcus torques (EPV76) B.faecis (EPV15) Blautia wexlerae SJTU1416 (EPV52) B. obeum (EPV20)Blautia WAL14507 (EPV64) B. producta (EPV21) Uncultured bacteriumSJTU1416 (EPV51) B. coccoides (EPV22) Uncultured bacterium GQ8980099(EPV47) B. hydrogenotrophica Eubacterium rectale (EPV35) (EPV23) B.hansenii (EPV24)

TABLE 7 Exemplary Prebiotics/Carbon Sources Chemical MoA L-ArabinoseC-Source, carbohydrate N-Acetyl-D-Glucosamine C-Source, carbohydrateD-Saccharic acid C-Source, carboxylic acid Succinic acid C-Source,carboxylic acid D-Galactose C-Source, carbohydrate L-Aspartic acidC-Source, amino acid L-Proline C-Source, amino acid D-Alanine C-Source,amino acid D-Trehalose C-Source, carbohydrate D-Mannose C-Source,carbohydrate Dulcitol C-Source, carbohydrate D-Serine C-Source, aminoacid D-Sorbitol C-Source, carbohydrate Glycerol C-Source, carbohydrateL-Fucose C-Source, carbohydrate D-Glucuronic acid C-Source, carboxylicacid D-Gluconic acid C-Source, carboxylic acid DL-a-Glycerol PhosphateC-Source, carbohydrate D-Xylose C-Source, carbohydrate L-Lactic acidC-Source, carboxylic acid Formic acid C-Source, carboxylic acidD-Mannitol C-Source, carbohydrate L-Glutamic acid C-Source, amino acidD-Glucose-6-Phosphate C-Source, carbohydrate D-Galactonic acid-g-LactoneC-Source, carboxylic acid DL-Malic acid C-Source, carboxylic acidD-Ribose C-Source, carbohydrate Tween 20 C-Source, fatty acid L-RhamnoseC-Source, carbohydrate D-Fructose C-Source, carbohydrate Acetic acidC-Source, carboxylic acid a-D-Glucose C-Source, carbohydrate MaltoseC-Source, carbohydrate D-Melibiose C-Source, carbohydrate ThymidineC-Source, carbohydrate L-Asparagine C-Source, amino acid D-Aspartic acidC-Source, amino acid D-Glucosaminic acid C-Source, carboxylic acid1,2-Propanediol C-Source, alcohol Tween 40 C-Source, fatty acida-Ketoglutaric acid C-Source, carboxylic acid a-Ketobutyric acidC-Source, carboxylic acid a-Methyl-D-Galactoside C-Source, carbohydratea-D-Lactose C-Source, carbohydrate Lactulose C-Source, carbohydrateSucrose C-Source, carbohydrate Uridine C-Source, carbohydrateL-Glutamine C-Source, amino acid m-Tartaric acid C-Source, carboxylicacid D-Glucose-1-Phosphate C-Source, carbohydrate D-Fructose-6-PhosphateC-Source, carbohydrate Tween 80 C-Source, fatty acid a-Hydroxyglutaricacid-g-Lactone C-Source, carboxylic acid a-Hydroxybutyric acid C-Source,carboxylic acid b-Methyl-D-Glucoside C-Source, carbohydrate AdonitolC-Source, carbohydrate Maltotriose C-Source, carbohydrate2′-Deoxyadenosine C-Source, carbohydrate Adenosine C-Source,carbohydrate Gly-Asp C-Source, amino acid Citric acid C-Source,carboxylic acid m-Inositol C-Source, carbohydrate D-Threonine C-Source,amino acid Fumaric acid C-Source, carboxylic acid Bromosuccinic acidC-Source, carboxylic acid Propionic acid C-Source, carboxylic acid Mucicacid C-Source, carboxylic acid Glycolic acid C-Source, carboxylic acidGlyoxylic acid C-Source, carboxylic acid D-Cellobiose C-Source,carbohydrate Inosine C-Source, carbohydrate Gly-Glu C-Source, amino acidTricarballylic acid C-Source, carboxylic acid L-Serine C-Source, aminoacid L-Threonine C-Source, amino acid L-Alanine C-Source, amino acidAla-Gly C-Source, amino acid Acetoacetic acid C-Source, carboxylic acidN-Acetyl-D-Mannosamine C-Source, carbohydrate Mono-MethylsuccinateC-Source, carboxylic acid Methylpyruvate C-Source, ester D-Malic acidC-Source, carboxylic acid L-Malic acid C-Source, carboxylic acid Gly-ProC-Source, amino acid p-Hydroxyphenyl Acetic acid C-Source, carboxylicacid m-Hydroxyphenyl Acetic acid C-Source, carboxylic acid TyramineC-Source, amine D-Psicose C-Source, carbohydrate L-Lyxose C-Source,carbohydrate Glucuronamide C-Source, amide Pyruvic acid C-Source,carboxylic acid L-Galactonic acid-g-Lactone C-Source, carboxylic acidD-Galacturonic acid C-Source, carboxylic acid Phenylethylamine C-Source,amine 2-Aminoethanol C-Source, alcohol Negative Control C-Source,negative control Chondroitin Sulfate C C-Source, polymer a-CyclodextrinC-Source, polymer b-Cyclodextrin C-Source, polymer g-CyclodextrinC-Source, polymer Dextrin C-Source, polymer Gelatin C-Source, polymerGlycogen C-Source, polymer Inulin C-Source, polymer Laminarin C-Source,polymer Mannan C-Source, polymer Pectin C-Source, polymerN-Acetyl-D-Galactosamine C-Source, carbohydrate N-Acetyl-Neuraminic acidC-Source, carboxylic acid b-D-Allose C-Source, carbohydrate AmygdalinC-Source, carbohydrate D-Arabinose C-Source, carbohydrate D-ArabitolC-Source, carbohydrate L-Arabitol C-Source, carbohydrate ArbutinC-Source, carbohydrate 2-Deoxy-D-Ribose C-Source, carbohydratei-Erythritol C-Source, carbohydrate D-Fucose C-Source, carbohydrate3-O-b-D-Galactopyranosyl-D-Arabinose C-Source, carbohydrate GentiobioseC-Source, carbohydrate L-Glucose C-Source, carbohydrate D-LactitolC-Source, carbohydrate D-Melezitose C-Source, carbohydrate MaltitolC-Source, carbohydrate a-Methyl-D-Glucoside C-Source, carbohydrateb-Methyl-D-Galactoside C-Source, carbohydrate 3-Methylglucose C-Source,carbohydrate b-Methyl-D-Glucuronic acid C-Source, carboxylic acida-Methyl-D-Mannoside C-Source, carbohydrate b-Methyl-D-XylosideC-Source, carbohydrate Palatinose C-Source, carbohydrate D-RaffinoseC-Source, carbohydrate Salicin C-Source, carbohydrate SedoheptulosanC-Source, carbohydrate L-Sorbose C-Source, carbohydrate StachyoseC-Source, carbohydrate D-Tagatose C-Source, carbohydrate TuranoseC-Source, carbohydrate Xylitol C-Source, carbohydrateN-Acetyl-D-Glucosaminitol C-Source, carbohydrate g-Amino-N-Butyric acidC-Source, carboxylic acid d-Amino Valeric acid C-Source, carboxylic acidButyric acid C-Source, carboxylic acid Capric acid C-Source, carboxylicacid Caproic acid C-Source, carboxylic acid Citraconic acid C-Source,carboxylic acid Citramalic acid C-Source, carboxylic acid D-GlucosamineC-Source, carbohydrate 2-Hydroxybenzoic acid C-Source, carboxylic acid4-Hydroxybenzoic acid C-Source, carboxylic acid b-Hydroxybutyric acidC-Source, carboxylic acid g-Hydroxybutyric acid C-Source, carboxylicacid a-Keto-Valeric acid C-Source, carboxylic acid Itaconic acidC-Source, carboxylic acid 5-Keto-D-Gluconic acid C-Source, carboxylicacid D-Lactic acid Methyl Ester C-Source, ester Malonic acid C-Source,carboxylic acid Melibionic acid C-Source, carbohydrate Oxalic acidC-Source, carboxylic acid Oxalomalic acid C-Source, carboxylic acidQuinic acid C-Source, carboxylic acid D-Ribono-1,4-Lactone C-Source,carboxylic acid Sebacic acid C-Source, carboxylic acid Sorbic acidC-Source, carboxylic acid Succinamic acid C-Source, carboxylic acidD-Tartaric acid C-Source, carboxylic acid L-Tartaric acid C-Source,carboxylic acid Acetamide C-Source, amide L-Alaninamide C-Source, amideN-Acetyl-L-Glutamic acid C-Source, amino acid L-Arginine C-Source, aminoacid Glycine C-Source, amino acid L-Histidine C-Source, amino acidL-Homoserine C-Source, amino acid Hydroxy-L-Proline C-Source, amino acidL-Isoleucine C-Source, amino acid L-Leucine C-Source, amino acidL-Lysine C-Source, amino acid L-Methionine C-Source, amino acidL-Ornithine C-Source, amino acid L-Phenylalanine C-Source, amino acidL-Pyroglutamic acid C-Source, amino acid L-Valine C-Source, amino acidD,L-Carnitine C-Source, carboxylic acid sec-Butylamine C-Source, amineD,L-Octopamine C-Source, amine Putrescine C-Source, amineDihydroxyacetone C-Source, alcohol 2,3-Butanediol C-Source, alcohol2,3-Butanedione C-Source, alcohol 3-Hydroxy-2-butanone C-Source, alcohol

TABLE 8 Bacterial Species Detected at Low Frequency in Vaginal Samplesfrom Vancomycin-Treated Mice Mean Median abundance day 6 abundance day 6Site Group Taxonomy (out of 10,000) (out of 10,000) vaginal VancomycinKF008552.1.1432 0.291242675 0.024255713 D_0_Bacteria;D_1_Proteobacteria; D_2_Gammaproteobacteria; D_3_Enterobacteriales;D_4_Enterobacteriaceae; D_5_Klebsiella; D_6_Klebsiella pneumoniaevaginal Vancomycin AB740357.1.1462 1.436524722 0 D_0_Bacteria;D_1_Proteobacteria; D_2_Gammaproteobacteria; D_3_Enterobacteriales;D_4_Enterobacteriaceae; D_5_Pantoea; D_6_Pantoea sp. NCCP-532 vaginalVancomycin DQ799428.1.1372 0.348310693 0 D_0_Bacteria;D_1_Verrucomicrobia; D_2_Verrucomicrobiae; D_3_Verrucomicrobiales;D_4_Verrucomicrobiaceae; D_5_Akkermansia; D_6_uncultured bacteriumvaginal Vancomycin JX094996.1.1390 0.348310693 0 D_0_Bacteria;D_1_Firmicutes; D_2_Clostridia; D_3_Clostridiales; D_4_Lachnospiraceae;D_5_Blautia; D_6_uncultured bacterium vaginal Vancomycin EU459716.1.12860.348310693 0 D_0_Bacteria; D_1_Firmicutes; D_2_Clostridia;D_3_Clostridiales; D_4_Lachnospiraceae; D_5_uncultured; D_6_unculturedbacterium vaginal Vancomycin EU457230.1.1391 0.696621386 0 D_0_Bacteria;D_1_Firmicutes; D_2_Clostridia; D_3_Clostridiales; D_4_Lachnospiraceae;D_5_Incertae Sedis; D_6_uncultured bacterium vaginal VancomycinEU459317.1.1373 0.348310693 0 D_0_Bacteria; D_1_Firmicutes;D_2_Clostridia; D_3_Clostridiales; D_4_Clostridiaceae 1; D_5_Clostridiumsensu stricto 1; D_6_uncultured bacterium vaginal VancomycinHM817954.1.1353 0.348310693 0 D_0_Bacteria; D_1_Firmicutes;D_2_Clostridia; D_3_Clostridiales; D_4_Lachnospiraceae; D_5_Roseburia;D_6_uncultured bacterium vaginal Vancomycin GQ134873.1.1373 0.3483106930 D_0_Bacteria; D_1_Firmicutes; D_2_Clostridia; D_3_Clostridiales;D_4_Clostridiaceae 1; D_5_Clostridium sensu stricto 1; D_6_unculturedbacterium vaginal Vancomycin FJ879074.1.1494 0.348310693 0 D_0_Bacteria;D_1_Firmicutes; D_2_Clostridia; D_3_Clostridiales; D_4_Lachnospiraceae;D_5_uncultured; D_6_uncultured bacterium vaginal VancomycinEU774816.1.1381 0.348310693 0 D_0_Bacteria; D_1_Firmicutes;D_2_Clostridia; D_3_Clostridiales; D_4_Clostridiaceae 1; D_5_Clostridiumsensu stricto 1; D_6_uncultured bacterium vaginal VancomycinEU775614.1.1398 0.417063419 0 D_0_Bacteria; D_1_Proteobacteria;D_2_Gammaproteobacteria; D_3_Enterobacteriales; D_4_Enterobacteriaceae;D_5_Enterobacter; D_6_uncultured bacterium

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to beobtained. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

The foregoing description of the embodiments of the invention has beenpresented for the purpose of illustration; it is not intended to beexhaustive or to limit the invention to the precise forms disclosed.Persons skilled in the relevant art can appreciate that manymodifications and variations are possible in light of the abovedisclosure.

Finally, the language used in the specification has been principallyselected for readability and instructional purposes, and it may not havebeen selected to delineate or circumscribe the inventive subject matter.It is therefore intended that the scope of the invention be limited notby this detailed description, but rather by any claims that issue on anapplication based hereon. Accordingly, the disclosure of the embodimentsof the invention is intended to be illustrative, but not limiting, ofthe scope of the invention, which is set forth in the following claims.

1. A method of increasing the duration of survival of a subjectreceiving a bone marrow transplant, comprising administering to thesubject a probiotic composition comprising an isolated, human-derivedbacterial population, such that the duration of survival of the subjectis increased.
 2. The method of claim 1, wherein administration of theprobiotic composition reduces the likelihood that the subject willdevelop sepsis following the bone marrow transplant.
 3. The method ofclaim 1, wherein administration of the probiotic composition reduces thelikelihood that the subject will develop graft versus host disease(GVHD) following the bone marrow transplant.
 4. A method of increasingthe duration of survival of a subject receiving a bone marrowtransplant, comprising: administering to the subject a probioticcomposition comprising an isolated bacterial population and apharmaceutically acceptable excipient, wherein the probiotic compositionreduces intestinal permeability in the subject; and administering to thesubject a prebiotic that enhances the activity of the bacterialpopulation, such that the duration of survival of the subject isincreased.
 5. A method of preventing graft versus host disease (GVHD) ina subject receiving a transplant, comprising administering to thesubject a probiotic composition comprising an isolated, human-derivedbacterial population, such that GVHD is prevented.
 6. The method ofclaim 5, wherein the subject is receiving a hematopoietic stem celltransplant.
 7. The method of claim 5, wherein the subject is receiving abone marrow transplant.
 8. The method of claim 5, wherein the subject isreceiving a solid organ transplant.
 9. The method of claim 8, whereinthe solid organ transplant is selected from the group consisting of akidney transplant, a heart transplant, a lung transplant, a skintransplant, a liver transplant, a pancreas transplant, an intestinaltransplant, an endocrine gland transplant, a bladder transplant, and askeletal muscle transplant.
 10. A method of reducing inflammation in thegastrointestinal tract of a subject receiving a transplant, comprisingadministering to the subject a probiotic composition comprising anisolated, anti-inflammatory bacterial population, such that inflammationin the gastrointestinal tract of the subject receiving the transplant isreduced.
 11. A method of reducing intestinal permeability in a subjectreceiving a transplant, comprising administering to the subject aprobiotic composition comprising an isolated bacterial population, suchthat the intestinal permeability of the subject receiving the transplantis reduced.
 12. A pharmaceutical composition comprising an isolatedanti-inflammatory bacterial population capable of decreasing secretionof a pro-inflammatory cytokine and/or increasing secretion of ananti-inflammatory cytokine by human peripheral blood mononuclear cells(PBMCs), and a pharmaceutically acceptable excipient.
 13. Thepharmaceutical composition of claim 12, further comprising a prebiotic.14. The method or composition of claim 1, wherein the bacterialpopulation comprises an isolated population of bacteria selected fromthe group consisting of Acidaminococcus intestine, Acinetobacterbaumannii, Acinetobacter lwoffii, Akkermansia muciniphila, Alistipesputredinis, Alistipes shahii, Anaerostipes hadrus, Anaerotruncuscolihominis, Bacteroides caccae, Bacteroides cellulosilyticus,Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii,Bacteroides fragilis, Bacteroides massiliensis, Bacteroides ovatus,Bacteroides salanitronis, Bacteroides salyersiae, Bacteroides sp. 1_1_6,Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:G0898099.1), Blautia uncultured PAC000178 s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof. 15-136. (canceled)
 137. The method of claim 4,wherein the bacterial population comprises an isolated population ofbacteria selected from the group consisting of Acidaminococcusintestine, Acinetobacter baumannii, Acinetobacter lwoffii, Akkermansiamuciniphila, Alistipes putredinis, Alistipes shahii, Anaerostipeshadrus, Anaerotruncus colihominis, Bacteroides caccae, Bacteroidescellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroidesovatus, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroidessp. 1_1_6, Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1), Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof.
 138. The method of claim 5, wherein thebacterial population comprises an isolated population of bacteriaselected from the group consisting of Acidaminococcus intestine,Acinetobacter baumannii, Acinetobacter lwoffii, Akkermansia muciniphila,Alistipes putredinis, Alistipes shahii, Anaerostipes hadrus,Anaerotruncus colihominis, Bacteroides caccae, Bacteroidescellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroidesovatus, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroidessp. 1_1_6, Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1), Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof.
 139. The method of claim 10, wherein thebacterial population comprises an isolated population of bacteriaselected from the group consisting of Acidaminococcus intestine,Acinetobacter baumannii, Acinetobacter lwoffii, Akkermansia muciniphila,Alistipes putredinis, Alistipes shahii, Anaerostipes hadrus,Anaerotruncus colihominis, Bacteroides caccae, Bacteroidescellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroidesovatus, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroidessp. 1_1_6, Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1), Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof.
 140. The method of claim 11, wherein thebacterial population comprises an isolated population of bacteriaselected from the group consisting of Acidaminococcus intestine,Acinetobacter baumannii, Acinetobacter lwoffii, Akkermansia muciniphila,Alistipes putredinis, Alistipes shahii, Anaerostipes hadrus,Anaerotruncus colihominis, Bacteroides caccae, Bacteroidescellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroidesovatus, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroidessp. 1_1_6, Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1), Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof.
 141. The pharmaceutical composition of claim12, wherein the bacterial population comprises an isolated population ofbacteria selected from the group consisting of Acidaminococcusintestine, Acinetobacter baumannii, Acinetobacter lwoffii, Akkermansiamuciniphila, Alistipes putredinis, Alistipes shahii, Anaerostipeshadrus, Anaerotruncus colihominis, Bacteroides caccae, Bacteroidescellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides fragilis, Bacteroides massiliensis, Bacteroidesovatus, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroidessp. 1_1_6, Bacteroides sp. 3_1_23, Bacteroides sp. D20, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium faecale, Bifidobacterium kashiwanohense,Bifidobacterium longum subsp. longum, Bifidobacterium pseudocatenulatum,Bifidobacterium stercoris, Blautia (Ruminococcus) coccoides, Blautiafaecis, Blautia glucerasea, Blautia (Ruminococcus) hansenii, Blautiahydrogenotrophica (Ruminococcus hydrogenotrophicus), Blautia(Ruminococcus) luti, Blautia (Ruminococcus) obeum, Blautia producta(Ruminococcus productus), Blautia (Ruminococcus) schinkii, Blautiastercoris, Blautia uncultured bacterium clone BKLE_a03_2 (GenBank:EU469501.1), Blautia uncultured bacterium clone SJTU_B_14_30 (GenBank:EF402926.1), Blautia uncultured bacterium clone SJTU_C_14_16 (GenBank:EF404657.1), Blautia uncultured bacterium clone S1-5 (GenBank:GQ898099.1), Blautia uncultured PAC000178_s(www.ezbiocloud.net/eztaxon/hierarchy?m=browse&k=PAC000178&d=2), Blautiawexlerae, Candidatus Arthromitus sp. SFB-mouse-Yit, Catenibacteriummitsuokai, Clostridiaceae bacterium (Dielma fastidiosa) JC13,Clostridiales bacterium 1_7_47FAA, Clostridium asparagiforme,Clostridium bolteae, Clostridium clostridioforme, Clostridiumglycyrrhizinilyticum, Clostridium (Hungatella) hathewayi, Clostridiumhistolyticum, Clostridium indolis, Clostridium leptum, Clostridium(Tyzzerella) nexile, Clostridium perfringens, Clostridium(Erysipelatoclostridium) ramosum, Clostridium scindens, Clostridium sp.14774, Clostridium sp. 7_3_54FAA, Clostridium sp. HGF2, Clostridiumsymbiosum, Collinsella aerofaciens, Collinsella intestinalis,Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Doreaformicigenerans, Dorea longicatena, Enterococcus faecalis, Enterococcusfaecium, Erysipelotrichaceae bacterium 3_1_53, Escherichia coli,Escherichia coli S88, Eubacterium eligens, Eubacterium fissicatena,Eubacterium ramulus, Eubacterium rectale, Faecalibacterium prausnitzii,Flavonifractor plautii, Fusobacterium mortiferum, Fusobacteriumnucleatum, Holdemania filiformis, Hydrogenoanaerobacteriumsaccharovorans, Klebsiella oxytoca, Lachnospiraceae bacterium3_1_57FAA_CT1, Lachnospiraceae bacterium 7_1_58FAA, Lachnospiraceaebacterium 5_1_57FAA, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus ruminis, Lactococcus casei, Odoribacter splanchnicus,Oscillibacter valericigenes, Parabacteroides gordonii, Parabacteroidesjohnsonii, Parabacteroides merdae, Pediococcus acidilactici,Peptostreptococcus asaccharolyticus, Propionibacterium granulosum,Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus faecis,Ruminococcus gnavus, Ruminococcus sp. ID8, Ruminococcus torques, Slackiapiriformis, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus cristatus, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus infantis, Streptococcus oralis, Streptococcus sanguinis,Streptococcus viridans, Streptococcus thermophiles, Veillonella dispar,and combinations thereof.